Isolation of primary mouse retinal ganglion cells using immunopanning-magnetic separation

Hong, Samin,Iizuka, Yoko,Kim, Chan Yun,Seong, Gong Je Molecular Vision 2012 Molecular vision Vol.18 No.-

<P><B>Purpose</B></P><P>To establish an effective system for isolating primary retinal ganglion cells (RGCs) from newborn mice.</P><P><B>Methods</B></P><P>The retinas were separated from enucleated eyeballs of Crl:CD-1 mice on postnatal day 1 to 4. RGCs were purified using three different methods, including two-step immunopanning (TSI), direct magnetic separation (DMS), and immunopanning-magnetic separation (IMS). Harvested cells were maintained for 24 h in a defined medium and then examined with immunocytochemistry, western immunoblotting, and real-time reverse transcription polymerase chain reaction (RT-PCR) for glial cell–specific glial fibrillary acidic protein (GFAP) and amacrine cell-specific syntaxin 1.</P><P><B>Results</B></P><P>As determined with immunofluorescence staining, RGCs purified by TSI were sparsely mixed with GFAP-positive astrocytes, and RGCs isolated by DMS were frequently mixed with syntaxin 1-positive amacrine cells. However, RGCs collected by IMS were seldom contaminated by GFAP-positive or syntaxin 1-positive cells. On western immunoblots, TSI cells showed significant GFAP expression, and DMS cells showed apparent syntaxin 1 expression, but IMS cells did not. Results of the real-time RT–PCR showed a similar tendency to those of the immunocytochemistry and western immunoblots.</P><P><B>Conclusion</B></P><P>Primary mouse RGCs were highly purified by the IMS method, combining the benefits of the TSI and DMS methods. This isolation method may provide a good experimental system for studying glaucoma in vitro.</P>

Original Article : The Neuroprotective Effect of Maltol against Oxidative Stress on Rat Retinal Neuronal Cells

( Yookyung Song ),( Samin Hong ),( Yoko Iizuka ),( Chan Yun Kim ),( Gong Je Seong ) 대한안과학회 2015 Korean Journal of Ophthalmology Vol.29 No.1

Purpose: Maltol (3-hydroxy-2-methyl-4-pyrone), formed by the thermal degradation of starch, is found in coffee, caramelized foods, and Korean ginseng root. This study investigated whether maltol could rescue neuroretinal cells from oxidative injury in vitro. Methods: R28 cells, which are rat embryonic precursor neuroretinal cells, were exposed to hydrogen peroxide (H2O2, 0.0 to 1.5 mM) as an oxidative stress with or without maltol (0.0 to 1.0 mM). Cell viability was monitored with the lactate dehydrogenase assay and apoptosis was examined by the terminal deoxynucleotide transferase- mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. To investigate the neuroprotective mechanism of maltol, the expression and phosphorylation of nuclear factor-kappa B (NF- κB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were evaluated by Western immunoblot analysis. Results: R28 cells exposed to H2O2 were found to have decreased viability in a dose- and time-dependent manner. However, H2O2-induced cytotoxicity was decreased with the addition of maltol. When R28 cells were exposed to 1.0 mM H2O2 for 24 hours, the cytotoxicity was 60.69 ± 5.71%. However, the cytotoxicity was reduced in the presence of 1.0 mM maltol. This H2O2-induced cytotoxicity caused apoptosis of R28 cells, characterized by DNA fragmentation. Apoptosis of oxidatively-stressed R28 cells with 1.0 mM H2O2 was decreased with 1.0 mM maltol, as determined by the TUNEL method. Western blot analysis showed that treatment with maltol reduced phosphorylation of NF-κB, ERK, and JNK, but not p38. The neuroprotective effects of maltol seemed to be related to attenuated expression of NF-κB, ERK, and JNK. Conclusions: Maltol not only increased cell viability but also attenuated DNA fragmentation. The results obtained here show that maltol has neuroprotective effects against hypoxia-induced neuroretinal cell damage in R28 cells, and its effects may act through the NF-κB and mitogen-activated protein kinase signaling pathways.

Transforming growth factor-β Does Not Induce Endothelin-1 Secretion in Primary Cultured Human Tenon’s Fibroblasts

홍사민,김찬윤,이종복,Iizuka Yoko,성공제 대한안과학회 2008 Korean Journal of Ophthalmology Vol.22 No.4

Subconjunctival fibrosis is closely related to scarring after glaucoma filtering surgery, and transforming growth factor-β (TGF-β) is known to play a crucial role in this scarring process. It induces the transformation of fibroblasts to myofibloblasts and the production of extracellular matrix (ECM) in the Tenon’s capsule. Endothelin-1 (ET-1) is thought to be secreted by fibroblasts and contributes to fibrosis in such stressed conditions. However, evidence regarding autocrine production of ET-1 from TGF-βstimulated human Tenon’s fibroblasts are lacking. In the present study, the authors evaluated the effects of TGF-β1 on ET-1 expression from primary cultured human Tenon’s fibroblasts. Our results demonstrate that TGF-β1 seemingly has no influence on the synthesis of ET-1 in human Tenon’s fibroblasts. Even though fibroblasts usually are not a major source of ET-1, these different cellular responses may implicate the characteristics of Tenon’s fibroblasts as being different to fibroblasts from other tissue. Studies concerning other intracellular signaling pathways associated with pro- and anti- fibrosis are also needed to confirm the similarities of fibroblasts from human Tenon’s capsule with that from other organs.

Original Articles : The Role of Focal Adhesion Kinase in the TGF-β-Induced Myofibroblast Transdifferentiation of Human Tenon`s Fibroblasts

( Sa Min Hong ),( Jong Bok Lee ),( Yoko Iizuka ),( Yoo Kyung Song ),( Gong Je Seong ),( Sueng Han Han ) 대한안과학회 2012 Korean Journal of Ophthalmology Vol.26 No.1

Purpose: To investigate the role of focal adhesion kinase (FAK) in transforming growth factor (TGF)-β-induced myofibroblast transdifferentiation of human Tenon`s fibroblasts. Methods: Primary cultured human Tenon`s fibroblasts were exposed to TGF-β1 for up to 48 hours. The mRNA levels of FAK, α smooth muscle actin (αSMA), and β-actin were determined by quantitative real time reverse transcription polymerase chain reaction. The protein levels of collagen type I, FAK, phospho-FAK, αSMA, and β-actin were determined by Western immunoblots. After the small interfering RNA targeting FAK (siRNAFAK) molecules were delivered into the cells, the expressions of αSMA proteins were determined by Western immunoblots. Results: In human Tenon`s fibroblasts, TGF-β1 significantly increased the mRNA and protein expressions of αSMA. However, when the action of FAK was inhibited using siRNAFAK, the TGF-β1-induced expression of αSMA was attenuated. Conclusions: Our data suggest that FAK may be associated with the TGF-β1-induced transdifferentiation of human Tenon`s fibroblasts to myofibroblasts, which is the essential step of subconjunctival fibrosis.

인간 섬유아세포에 대한 마이토마이신 C와 피르페니돈의 독성과 항섬유화효과

박경수,홍사민,이주카요코,김찬윤,성공제.Kyoung Soo Park. MD. Sa Min Hong. MD. Yoko Iizuka. MD. PhD. Chan Yun Kim. MD. Gong Je Seong. MD 대한안과학회 2014 대한안과학회지 Vol.55 No.7

<b>Purpose:</b> The cytotoxicities and anti-fibrotic effects of mitomycin C and pirfenidone on human dermal fibroblast were evaluated. <b>Methods:</b> Initially, 24-hour cell cultures were exposed to transforming growth factor (TGF)-β1, different concentrations of mitomycin C, and pirfenidone solutions in order to evaluate cytotoxicity. Expressions of fibronectin, collagen type 1, α smooth muscle, and β-actin were evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot in mitomycin C solutions at concentrations of 4 μg/mL and 20 μg/mL, and in pirfenidone solutions at 250 μg/mL and 500 μg/mL. <b>Results:</b> In comparison to cell cultures exposed to TGF-β1 solutions, cytotoxicities were increased in solutions of mitomycin C at 4 μg/mL, 20 μg/mL, 40 μg/mL and pirfenidone at 500 μg/mL, 750 μg/mL, 1,000 μg/mL (<em>p</em> < 0.05, Mann Whitney U-test). The results of real-time RT-PCR show that expressions of fibronectin, collagen type 1, and α smooth muscle were significantly more decreased in all concentrations of mitomycin C and pirfenidone compared to those in TGF-β1 solution. In western blot analysis, expressions of fibronectin and α smooth muscle were decreased in all concentrations of mitomycin C and pirfenidone compared to TGF-β1 solution. <b>Conclusions:</b> Both drugs have cytotoxicities and anti-fibrotic effects, but pirfenidone was found to have less cytotoxicity and mitomycin C was found to have more anti-fibrotic effects when compared to each other. J Korean Ophthalmol Soc 2014;55(7):1077-1083