• Journal of Genetic Medicine
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• v.12 no.1
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• pp.25-30
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• 2015

Purpose: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. Materials and Methods: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. Results: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. Conclusion: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.

• The Korean Journal of Pharmacology
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• v.30 no.2
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• pp.167-179
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• 1994

Recent evidence indicates that glial cells have a wide range of funtions which are critical for maintaining a balanced homeostatic environment in the central nervous system(CNS) peripheral nervous system(PNS). Morever, astrocytes are known to participate in the tissue repair and neuroimmunologic events within the CNS through many kinds of growth factors and cytokines. We investigated the effect of $TGF\;{\beta}_1$, on the growth and biochemical changes of rat glial cells in culture. The proliferative effect was determined by $^3H-thymidine$ uptake and the double immunostain with anti-cell-specific marker and anti-Bromodeoxyuridine(BrdU) antibody. To check the effect of biochemical changes we compared the amounts of glial fibrillar acidic protein(GFAP) and the activity of glutamine synthetase(GS) in astrocyte. And the amounts of myelin basic protein and the activity of 2',3'-cyclic nucleotide phosphohydrolase(CNPase) were measured in oligodendrocyte and the amounts of peripheral myelin in Schwann cell. When $TGF\;{\beta}_1$, was treated for 2 days with cultured glial cell, $TGF\;{\beta}_1$, decreased the $^3H-thymidine$ uptake and proliferation index of double immunostain of astrocytes, which indicates the inhibition of astroglial DNA synthesis, but stimulated the growth of Schwann cell. Also, $TGF\;{\beta}_1$, decrease the GS activity and increased the amounts of GFAP in astrocyte. In the case of Schwann cells the amounts of peripheral myelin was increased when treated with $TGF\;{\beta}_1$. However, $TGF\;{\beta}_1$, didn't show any effect on the proliferation and biochemical changes in oligodendrocyte. These results suggest that $TGF\;{\beta}_1$, might have a critical action in the regulation of proliferation and biochemical changes in glial cells, especially astrocyte.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2019.05a
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• pp.528-532
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• 2019

The dorsal root ganglion (DRG) was isolated from mouse embryos and Schwann cells and neuronal cells were cultured in vitro. The neurons and Schwann cells were cultured separately and the two kinds of cells were cultured together for three weeks. Generation of myelination was confirmed by transmission electron microscope and confocal microscope using a myelinaion protein, myelin protein zero (MPZ) antibody. The sindbis virus was infected for three days in the myelinated culture cells and then demyelination was carried out. The process of demyelination was also confirmed by transmission electron microscopy and confocal microscopy using myelin protein zero (MPZ) antibody. The study was supported by a Basic Research Program through the National Research Foundation (NRF) funded by the Ministry of Science and Technology, ICT and Future Plans (NRF-2016R1A2B4016552 and 2017R1A2B3005753).

• Applied Microscopy
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• v.18 no.1
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• pp.92-102
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• 1988

The ototoxic effects of salicylate on the ultrastructure of spiral ganglion cells were examined. Sodium salicylate($50{\sim}60\/kg$ body weight, once a day for 7 days) were injected subcutaneously to $5{\sim}6$ week-old fifteen Sprague-Dawley rats. Animals were sacrificed 24 hours (group 1), 6 weeks (group 2) or 10 weeks (group 3) after the last injection. In group 1 animals, distention of membranous cisternae was found in the cytoplasm of ganglion cells, satellite cells and Schwann cells in which enlargement or multicystic cytosome formation of the mitochondria were shown. In group 2 animals, membranous cisternae became larger or fused to form larger vacuoles or cysts. Shrinkage of spiral ganglion cell cytoplasm and loosening of myelin sheath were seen. In group 3 animals, extensive swelling or loss of nerve fibers were shown along with the folding or partial loss of myelin sheath which caused leakage of ganglion cell cytoplasm. It was concluded that the ototoxicity of salicy-late caused the ultrastructral changes of the spiral ganglion cells which became more severe in group 2 and 3 animals. The possibility of retrograde degeneration following the sensory cell changes was suggested.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2017.05a
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• pp.714-717
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• 2017

Schwann cells and Neuronal cells were isolated from dorsal root ganglion (DRG) in embryos of rat in vitro respectively. The cultured Schwann cells and cultured neuronal cells, respectively were co-cultured in a same plate. These cells were performed accomplishment of myelination. This myelinated co-culture system was infected by Semliki forest virus and then induced demyelination processing in this myelinated co-culture. We identified myelination and demyelination processing using antibody of peripheral myelin protein 22 (PMP 22) meaning presence of myelinated neuron.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2016.10a
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• pp.919-922
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• 2016

Neuronal cells and Schwann cells from dorsal root ganglion (DRG) in embryos of rat were isolated and cultured in vitro respectively. The purified neuronal cells added with anti-mitotic agents and purified Schwann cells were co-cultured and then accomplished myelination processing. This myelinated co-culture system was infected by herpes simplex virus-1 and then accomplished demyelination processing in this myelinated co-culture. We identified myelination and demyelination processing using antibody of neuropeptide Y meaning presence of myelinated neuron.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.5
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• pp.375-395
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• 2006

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2015.05a
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• pp.943-946
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• 2015

For myelination, Schwann cells and neuron cells from dorsal root ganglion (DRG) of rat embryos (E16) were cultured in vitro system. The purified DRG cells with anti-mitotic agents and purified Schwann cells were cocultured and then accomplished myelination processing. Treatment of M. leprae-specific phenolic glycolipid-1 (PGL-1) into this coculture system was performed and then accomplished demyelination. Therefore, we identified demyelination processing using antibody of myelin basic protein (MBP).

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2016.05a
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• pp.718-721
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• 2016

Schwann cells and neuron cells from dorsal root ganglion (DRG) in embryos of rat were cultured in vitro respectively. The purified neronal cells added with anti-mitotic agents and purified Schwann cells were cocultured and then accomplished myelination processing. Infection of Semliki forest virus into this myelinated co-culture system was performed and then accomplished demyelination. We identified myelination and demyelination processing using antibody of neuropeptide Y meaning presence of myelinated neuron.

• The Journal of Internal Korean Medicine
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• v.33 no.1
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• pp.83-101
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• 2012

Objectives : This study was aimed to investigate the therapeutic effect of Bogijetongtanggammi-bang (BJTG) on injury of the peripheral nerve tissues. Methods : Rats were divided into 2 groups. The rats of the first group were injected with Taxol (1.25 mg/kg) to their sciatic nerves, once each. The sciatic nerves of the rats of the second group were crushed by forcept for 30 seconds. Rats were administered with BJTG (400 mg/kg) or 0.9% saline for 5 days. Changes of DRG neurons, Schwann cells, Cdc2, caspase 3. phospho-p44/42 Erk1/2, phospho-vimentin and ${\beta}1$ integrin were observed by fluorescent microscope and analysed in western blot. Results : In Taxol-treated SD rat models, BJTG up-regulated neurite outgrowth, Schwann cells, Cdc2 and phospho-Erk1/2, and down-regulated caspase 3. In pressure-injured rat models, BJTG up-regulated axons of sciatic nerve, Schwann cells, Cdc2, phospho-vimentin, ${\beta}1$ integrin, and down-regulated caspase 3. Conclusions : Taken together, BJTG was promotive of nerve regeneration on SNI as well as Taxol-induced nerve injury. BJTG had a pharmaceutical property enhancing recovery of injured peripheral nerves and could be a candidate for drug development after further research.