• Radiation Oncology Journal
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• v.21 no.4
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• pp.315-321
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• 2003

Purpose: This study was peformed to Investigate apoptosis by radiation In the developing fetal rat brain. Materials and Methods: Fetal blains were Irradiated In utero between the 17th and 19th days of fetal life (El7-19) by linear accelerator. A dose of Irradiation ranging from 1 Gy to 4 Gy was used to evaluate dose dependency. To test time dependency the ra)s were Irradiated with 2 Gy and then the fetal brain specimens were removed at variable 41me course; 1, 3, 5, 12 and 24 hours after the onset of irradiation. Immunohistochemlcal staining using in situ 707-mediated dUTP nick end labelling (TUNEL) technlfue was used for apoptotic cells. The cerebral cortex, including three zones on coriicai zone (Cf). Intermediate zone (if), and ventricular zone (VZ), was examined. Results : TUNEL positive cells revealed typical features of apoptotic cells under light microscope In the fetal rat cerebral cortex. Apoptotic cells were not found In the cerebral cortex of non-Irradiated fetal rats, but did appear In the entire cerebral cortex after 1 Gy Irradiation, and were more expensive at the ventricular and Intermediate zones than at the cortical zone. The extent of apoptosis was Increased with Increasing doses of radiation. Apoptosis reached the peak at S hours after the onset of 2 Gy Irradiation and persisted until 24 hours. Conclusion: Typical morphological features of apoplosis by irradiation were observed In the developing fetal rat cerebral cortex. It was more extensive at the ventricular and Intermediate zones than at the cortical zone, which suggested that stem cells or early differentiated cells are more radiosensitive than differentiated cells of the cortical zone.

• Applied Microscopy
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• v.40 no.2
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• pp.81-88
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• 2010

The fine structural characteristics of the apoptotic cells in the cutaneous epithelium of the anuran tadpole of the black-spotted frog, Rana nigromaculata was examined using the TUNEL (Terminal deoxynucleotidyl transferase-mediated biotinylated d-Uridine triphosphate Nick End Labeling) staining technique and TEM (transmission electron microscopy) observations. The cutaneous epithelium of the tadpole was composed of stratified cuboidal cells and the apoptotic cell death was observed continuously during the tail degeneration stages from the Shumway stage number 31 to 33. The early apoptotic cells shown in the epithelium demonstrated condensation and margination of the chromatin material at the nuclear periphery, and nuclear breakdown and cytoplasmic condensation were followed. Subsequent cytoplasmic degeneration of the apoptotic cell were produced by membrane-bounded cell fragments with relatively well preserved organelles. Following the processes of autophagic degradation, the late apoptotic cells being phagocytosed by other surrounding cells. These nearby cells, presumptive intraepithelial macrophages, contain a variety of lysosomal residual bodies which fuses with other cell organelles or other cytoplasmatic material to form secondary lysosomes. They are soon transformed into lamellar shaped vesicles and finally disappeared during the process of degradation.

• Applied Microscopy
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• v.39 no.2
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• pp.107-115
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• 2009

The programed cell death of the cutaneous epithelial tissue during tail regression stages in anuran tadpoles of the blackspotted frog, Rana nigromaculata were visualized by the histochemical and transmission electron microscopic techniques. Metamorphotic changes in the tail regression during the period of the Shumway stage number 31 to 33 are characterized by the disappearance of mucous layer and formation of compound epithelium through cutaneous thickening. Following the TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated d-uridine triphosphate nick end labeling) staining technique, the apoptotic cells were detected at the distal region of the tail skin initially, but they can be seen at the proximal region according to their following development. It has been also revealed that the number of the TUNEL-positive cells gradually increased from apical to basal direction of the epithelial layers during the tail regressing stages. Following the TEM observation, the early apoptotic cells shown in the epithelium demonstrated condensation and margination of the chromatin material at the nuclear periphery. Another epithelial apoptotic cells were shown nuclear fragmentation, membrane blebbing and cytoplasmic condensation. Following the process of the apoptotic degradation, well preserved organelles and nuclear fragments can be identified in the cytoplasm of lysosome-rich cells, however they soon reduced to lysosomal residual bodies through the progressive degradation.

• Journal of Oral Medicine and Pain
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• v.38 no.4
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• pp.291-298
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• 2013

On this study, we treated rats with restraint stress, and observed the changes with an optical microscope. Within the salivary gland tissue, we measured cell apoptosis cycle evaluation which show positive reaction on TUNEL assay, and compared within the groups. For this study, 18 rats were divided into 3 groups; 1) 2 rats of group I were selected as a normal control. 2) 2 rats of group II, as a experimental control were placed in the restraint cone for 2 hours 3) 14 rats of group III were placed in the restraint cone for 2 hours once a day. The rats were sacrificed immediately (group II, as a experimental control), 1, 2, 3, 4, 5, 6 and 7days after application of the stress and the both parotid glands were excised. The conclusions follow. 1. 5 days after giving an confining stress to the parotid gland of Rats, we can observe the hypotropy and pus and inflammation of Rat parotid gland acinar cells, and after 7 days, we can see a cell apoptosis. 2. Through the In situ DNA end labeling assay and TUNEL dye, on serous glands, benign tumor cell increased with statistically significant result after 5 days from confining stress. And the index shows maximum value on 7th days, which is same result with histological opinion. Therefore, our study shows that a cell apoptosis can be induced by restraint stress on salivary gland tissue, and we think more study should be accomplished about the cell signaling pathway in the future.

• Journal of Korean Neurosurgical Society
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• v.38 no.3
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• pp.215-220
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• 2005

Objective : Many researchers believe that the hypothermia shows neuro-protective effect on brain injury. To understand the molecular mechanism of the hypothermic treatment, this study investigated its effects on the expression of cell death or survival related proteins such as p53, Bcl-2 and Bax in the rat traumatic brain injury[TBI] model. Methods : Twenty rats [Spraque Dawley, $200{\sim}250g$] were subjected to the brain injury of moderate severity [$2.4{\sim}2.6atm$] using the fluid percussion injury device and five rats were received only same surgery as controls. During 30minutes after the brain injury, the hypothermia group was maintained the body temperature around $34^{\circ}C$ while the control group were maintained that of $36^{\circ}C$. Five rats in each group were sacrificed 12h or 24h after brain injury and their brain sections was analyzed for physical damages by H-E stains and the extent of apoptosis by TUNEL assay and immunohistochemical stains. The tissue damage after TBI was mainly observed in the ipsilateral cortex and partly in the hippocampus. Results : Apoptosis was observed by TUNEL assay and the Bax protein was detected in both sample which harvested 12h and 24h after TBI. In the hypothermia treatment group, tissue damage and apoptosis were reduced in HE stains and TUNEL assay. In hypothermia treatment group rat shows more expression of the Bcl-2 protein and shows less expression of the Bax protein, at both 12h and 24h after TBI. Conclusion : These results show that the hypothermia treatment is an effective treatment after TBI, by reducing the apoptotic process. Therefore, it could be suggested that hypothermia has a high therapeutic value for treating tissue damages after TBI.

• Development and Reproduction
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• v.15 no.4
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• pp.373-383
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• 2011

Tributyltin (TBT) is one of endocrine disrupters which are known as having similar function to sex steroid hormone inducing apoptosis in various tissues of rodents. Recently, it has been reported that TBT induces apoptosis in thymus causing the decreased thymic function, but little is known about the mechanism. To elucidate the mechanism, three-week-old SD female rats were orally administrated with TBT 1, 10, and 25 mg per body weight (kg) and sesame oil as a control for 7 days. On day 8, the thymi were obtained and weighed, and then the number of thymocytes was counted. We also performed H&E staining, TUNEL assay, and Annexin V flow cytometric analysis to examine the apoptosis rates and the structure in the thymus. Next, we investigated the adipogenesis and apoptosis-related mRNA expression levels in the thymi by real-time PCR. The thymic weight and the number of thymocytes were decreased by TBT in a dose-dependent manner. As a result of the H&E staining, the boundary between cortical and medullary area was blurred in the thymi of TBT treated rats compared to those of controls. In the results of TUNEL assay and Annexin V flow cytometric analysis, apoptosis rates in the thymus were increased after TBT treatment. The expression levels of thymic epithelial cell marker genes such as EVA, KGF, AIRE, and IL-7 were significantly decreased in the thymi of TBT treated rats, but $PPAR{\gamma}$, aP2, PEPCK, and CD36 were significantly increased. The expression of $TNF{\alpha}$ and TNFR1 as apoptosis-related genes also was significantly increased after TBT treatment. The present study demonstrates that TBT can increase the expression of adipogenesis and apoptosis-related genes leading to apoptosis in the thymus. These results suggest that the increased adipogenesis of thymus by TBT exposure might induce apoptosis in the thymus resulting in a loss in thymic immune function.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.2
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• pp.617-622
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• 2014

Voacanga globosa (Blanco), a plant endemic to the Philippines, is traditionally used especially by indigenous people of Bataan in the treatment of ulcers, wounds and tumorous growths. This study aimed to provide scientific evidence to therapeutic properties by determining cytotoxic and pro-apoptotic activity of HPLC fractions from leaves on HCT116 human colon carcinoma and A549 human lung carcinoma cell lines. Ethanolic extraction was performed on V globosa leaves followed by hexane and ethyl acetate partitioning. Silica gel column chromatography and high performance liquid chromatography (HPLC) produced MP1, MP2 and MP3 fractions. Cytotoxic activity of the fractions was determined through MTT assay against the cancer cell lines HCT116 and A549 and the non-cancer AA8 Chinese hamster ovarian cell line. Pro-apoptotic activities of the most active fractions were further assessed through DAPI staining, TUNEL assay and JC-1 mitochondrial membrane potential assay with HCT116 cells. While the MPI fraction exerted no significant activity against all cell lines tested, MP2 and MP3 fractions demonstrated high toxicity against HCT116 and A549 cells. The MP3 fraction induced formation of apoptotic bodies, condensed DNA and other morphological changes consistent with apoptosis of HCT116 cells and TUNEL assay showed significant increase in DNA fragmentation over time. In these cells, the MP3 fraction also induced mitochondrial membrane destabilization, which is generally associated with the beginning of apoptosis. Phytochemical analysis demonstrated the presence only of saponins and terpenoids in the MP3 fraction. The results indicate that the MP3 fraction exerts cytotoxic activity on HCT116 cells via induction of apoptosis triggered by loss of mitochondrial membrane potential crucial for cell survival.

• Archives of Plastic Surgery
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• v.36 no.1
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• pp.11-18
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• 2009

Purpose: Excessive scarring in the forms of keloid and hypertrophic scar could be a consquence of the accumulation of granulation tissue cells due to aberrant control of apoptosis. Verapamil retard extracelluar matrix production and inhibits VEGF production in primary cultured keloid fibroblast. The object of this study was effect of verapamil on VEGF expression and apoptosis in early wound scarring of the rabbit ear. Methods: Full thickness wounds were created on the ventral side of 6 New Zealand rabbits's ear. 16 days after initial wounding verapamil and saline were injected each scars and scars were harvested 1 week, 2 weeks, 4 weeks later. The wounds were stained with hematoxylin and eosin, TUNEL stain, immunohistochemical stain for VEGF and calculated scar elevation index. Results: Histologic analaysis demonstrated significant reduction in inflammation, vascularity and improvement in dermal collagen organization in experimental group. In TUNEL staining apotosis positive cells were increased and immunohistochemial staining of VEGF demonstrated significant reduction of VEGF expression in experimental group. No significant difference was noted in scar elevation index between two groups. Conclusion: This study suggest that intralesional injection of verapamil on early wound scarring of the rabbit ear decreased VEGF production and increased apoptosis and have a benefit on the pathophysiology of scar formation.

• The Journal of Internal Korean Medicine
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• v.27 no.2
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• pp.316-326
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• 2006

Backgrounds & Objects: The aim of this study was to investigate the radioprotective effect of Shengmai-san(SMS), a herbal medicine, on mice jejunal crypt cell survival and Apoptosis. Methods: Mice were devided into 4 groups according to radiation dose and SMS treatment: Normal was the group without irradiation. Control was the group treated with D.W before 10 Gy irradiation. SMS 2.9 was sample group treated with 2.9 mg/10 g of SMS extract before 10 Gy irradiation and SMS 29 was sample group treated with 29 mg/10 g of SMS extract before 10 Gy irradiation. And Each group were sacrificedat 24 hours and 72 hours after irradiation. To analyze the crypt survival, hematoxylin-eosin staining was used and to analyze the apoptosis, the TUNEL assay was done. Results: 1. From the microcolony survival assay, the SMS 2.9 and SMS 29 showed the radioprotective effect with a statistical significance compared to the control group at 24 hr (P < 0.01) and 72 hr (p < 0.001) after 10 Gy irradiatien. And the differences of radioprotective effect between SMS 2.9 and SMS 29 were net significant. 2. The results of the TUNEL assay showed that the apoptotic index in SMS 2.9 and SMS 29 was significantly decreased, as compared to the control group at both 24 hr ( p < 0.01) and 72 hr (SMS 2.9 : p < 0.001. SMS 29 : P < 0.01) after 10Gy irradiation And the differences of between SMS 2.9 and SMS 29 were not significant. Conclusions: It could be suggested that the Shengmai-san has a prominent Protective effect in mice intestines against the radiation damage. And the radieprotective effect seems to be related to inhibition of the apoptosis.

• Applied Microscopy
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• v.42 no.2
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• pp.53-59
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• 2012

We evaluated the level of toxicity by LC50 and investigated the mechanism of brain impairment and GFAP expression by light and fluorescence microscopes in the pale chub, Zacco platypus, treated with fenvalerate. Survival rate was decreased according to the rise of fenvalerate concentration, and LC50 concentration was $27.79{\mu}g/L$. Apoptosis was increased according to the rise of fenvalerate concentration by TUNEL assay which determine apoptotic cell death population. Also, GFAP expression was increased in the periventricular zone. These results suggest that apoptosis might be a major mechanism to brain impairment of the pale chub by fenvalrerate. Increased GFAP expression in the periventricular zone would be an index of brain impairment. Taken together, this study might contribute to reveal the pathological mechanism of fish brain impairment by insecticide of pyrethroid, and to be an useful basic data for preservation of aquatic ecosystem.