• Biomedical Science Letters
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• v.8 no.3
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• pp.161-165
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• 2002

Apoptosis can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. Several techniques for the qualitative and quantitative detection of this process have been established; recently, an in situ nick end-labelling technique based on the detection of DNA fragmentation, which is a molecular characteristic of apoptotic cell death, was described. Applying this method to paraffin sections of rat tissues, sensitivity was observed to be inconsistently low with regard to the expected number of apoptotic cells. I describe a new modified method for formalin-fixed, paraffin-embedded tissue sections, pretense pretreatment to permeate the tissue sections that involves an TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is acknowledged as a method of choice in the rapid identification and quantification of the apoptotic cell fraction in paraffin tissue preparations. TUNEL was performed without apoptosis and with apopotosis samples to each of the three concentrations of proteinase K (10, 25, 40 mg/ml) pretreatments. In this study, I show that chemical pretreatments of the tissue sections in proteinase K (25 mg/ml for 15 min at room temperature) considerably enhances the sensitivity of this nick end labelling technique.

• Biomedical Science Letters
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• v.18 no.2
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• pp.112-122
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• 2012

A gradual change of molecules that are related in fission and fusion is occurred during aging process. Although aging effects on mitochondrial fusion and fission are investigated, it is still unclear that the extent of the change in mitochondria fusion and fission periodically. In this study we investigated the changes of mitochondrial proteins involved in fusion (Mfn2, Opa1) and fission (Drp1, Fis1) in the human gracilis muscle ranging from 10 to 50 years of age (n=40). The gracilis muscle showed a significant increase in muscle apoptotic changes in the age of 50s compared with 10s by using in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The expression levels of Drp1 and Fis1 (P<0.01, P<0.05) mRNA were significantly elevated and the Mfn2 and Opa1 (P<0.01, P<0.05) levels were decreased from older individuals. The ratio of fission and fusion was altered and the level of increment of fission gene was greater than fusion gene decrement in the age of 50s. These findings suggest that changes of mitochondrial fusion and fission proteins related with aging might contribute to aged muscle apoptosis.

• Korean Journal of Veterinary Service
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• v.35 no.2
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• pp.105-109
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• 2012

Apoptosis is a host defense mechanism that the cell uses to limit production of infectious pathogens. Although many bacteria, viruses and parasites can induce apoptosis in infected cells, some pathogens usually exhibit the ability to suppress the induction of apoptosis in the infected cells. Sophisticated evasion strategies of obligate intracellular parasites, in particular prevention of host cell apoptosis, are necessary to ensure successful replication. To study the ability of Eimeria tenella in this regard, in vitro experiments were performed applying Madin-Darby bovine kidney (MDBK) cells as host cell. We have demonstrated that productive infection of adherent cell lines by E. tenella resulted in an anti-apototic effect. This phenomenon was confirmed using in situ terminal deoxynucleotidyl transferase-mediated (TdT) deoxyuridine triphosphates (dUTP)-fluorescein nick end labeling (TUNEL) assay to detect apoptosis. Therefore, E. tenella could complete its cycle of productive infection while inducing anti-apoptosis in the infected cells. This finding might have implications for the pathobiology of E. tenella and other Eimeria species.

• The Journal of Korean Medicine
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• v.26 no.4
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• pp.12-21
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• 2005

Objectives: In the present study, we investigated whether an aqueous extract of Armeniacae semen induces apoptotic neuronal cell death upon mouse N2a neuroblastoma cells. Methods: 1. Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTI) assay. 2. For in situ detection of apoptotic cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, 4,6-diamidino-2-phenylindole (DAPI) staining. 3. The fraction of cells was revealed by flow cytometric analysis used that. 4. For detection of apoptotic DNA cleavage, DNA fragmentation assay was performed. 5. For detection of bax and bcl-2, Western blot analysis was performed. 6. Caspase enzyme activity was measured using caspase-3 assay. Results: From the present results, N2a neuroblastoma cells treated with Armeniacae semen extract exhibited several characteristics of apoptosis. A treatment of Armeniacae semen extract was shown to increase the expression of Bax, a proapoptotic protein, and the treatment decreased the expression of Blc2, an anti-apoptotic protein. In addition, Armeniacae semen extract increased the caspase-3 enzyme activity. Conclusions: The present results show that Armeniacae semen extract induces apoptotic cell death in mouse N2a neuroblastoma cells.

• Clinical and Experimental Pediatrics
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• v.64 no.8
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• pp.422-429
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• 2021

Background: Carnosine has antioxidative and neuroprotective properties against hypoxic-ischemic (HI) brain injury. Hypothermia is used as a therapeutic tool for HI encephalopathy in newborn infants with perinatal asphyxia. However, the combined effects of these therapies are unknown. Purpose: Here we investigated the effects of combined carnosine and hypothermia therapy on HI brain injury in neonatal rats. Methods: Postnatal day 7 (P7) rats were subjected to HI brain injury and randomly assigned to 4 groups: vehicle; carnosine alone; vehicle and hypothermia; and carnosine and hypothermia. Carnosine (250 mg/kg) was intraperitoneally administered at 3 points: immediately following HI injury, 24 hours later, and 48 hours later. Hypothermia was performed by placing the rats in a chamber maintained at 27℃ for 3 hours to induce whole-body cooling. Sham-treated rats were also included as a normal control. The rats were euthanized for experiments at P10, P14, and P35. Histological and morphological analyses, in situ zymography, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, and immunofluorescence studies were conducted to investigate the neuroprotective effects of the various interventional treatments. Results: Vehicle-treated P10 rats with HI injury showed an increased infarct volume compared to sham-treated rats during the triphenyltetrazolium chloride staining study. Hematoxylin and eosin staining revealed that vehicle-treated P35 rats with HI injury had decreased brain volume in the affected hemisphere. Compared to the vehicle group, carnosine and hypothermia alone did not result in any protective effects against HI brain injury. However, a combination of carnosine and hypothermia effectively reduced the extent of brain damage. The results of in situ zymography, TUNEL assays, and immunofluorescence studies showed that neuroprotective effects were achieved with combination therapy only. Conclusion: Carnosine and hypothermia may have synergistic neuroprotective effects against brain damage following HI injury.

• Imaging Science in Dentistry
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• v.31 no.4
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• pp.227-233
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• 2001

Purpose: The purpose of this study was to investigate the apoptosis induction in tissues constituting the craniofacial region of growing rat by irradiation. Materials and Methods: The submandibular gland, brain, articular cartilage of condylar head, and calvarium were extracted from 20-day-old rats irradiated 10 Gy. Apoptosis of each tissue was examined by DNA fragmentation and estimated quantitatively using apoptotic index on TUNEL assay. Apoptotic index of each tissue was calculated by the equation for apoptotic cells/total cells × 1,000 on the images of confocal laser scanning microscopy. Apoptotic index was analyzed statistically according to the time lapse after irradiation on the tissues. Results : In the submandibular gland, apoptotic index was significantly increased from 6 hours after irradiation showing the highest value at 12 hours and decreased to the control level at 3 days after irradiation. In the brain, apoptotic index was abruptly reached to the maximum value at 6 hours after irradiation and decreased to the control level at 4 days after irradiation. Articular cartilage and calvarium showed no or little apoptotic signals. The results obtained by the apoptotic index accorded with that of DNA fragmentation. Conclusion : Radiation was closely related with the apoptosis of submandibular gland and brain but, not related with the apoptosis of the articular cartilage of condylar head and calvarium. The changes induced by radiation of the hard tissues would not be explained by apoptosis.

• 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.

• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.4
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• pp.709-727
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• 2001

The pattern of programmed cell death(PCD) has been examined during the early developmental period of development in mouse embryos, from embryonic day 4.5(E4.5) to E11.5 Embryos from Balb/c breedings were harvested at various embryonic stages between E4.5 and El1.5. Cell death was analysed by in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) staining in tissue sections and whole embryos. At the blastocyst stage(E4.5), a very few apoptotic cells were found in the inner cell mass of the blastocyst. In the early egg cylinder stage(35.0-5.5), a few apoptotic cells were detected in the embryonic ectoderm, the embryonic endoerm and the proamniotic cavity. In the advanced egg cylinder stage(E5.5-6.5), TUNEL-posifive cells were observed in the extra-embryonic ectoderm and extra-embryonic endoderm as well as in the embryonic ectoderm, embryonic visceral endoderm and proamniotic cavity. In the streak stage(E6.75-7.75), many TUNEL-positive cells were found in the ectoplacental cone. In contrast, only very few apoptotic cells were found in the chorion and extra-embryonic endoderm in extra-embryonic regions. In intra-embryonic region, a few apoptotic cells were randomly found in the embryonic ectoderm, mesoderm and visceral endoderm. At the early somitogenesis stage(E8.0-8.5), most apoptotic cells were observed in the most cranial portion of neural fold (neural ectoderm and adjacent ectoderm). At the mid somitogenesis stage(39.0-9.5), the otic placode first showed TUNEL-positive at this stage. Small number of TUNEL-positive cells were also first seen around optic placode and branchial arches. Three streams of TUNEL-positive cells were clearly seen in the cranial region at 59.5-9.75. At E10.5, apoptotic cells were localized in the developing eye, the junctional portion of medial nasal, lateral nasal and maxillary processes, the lateral portion of branchial arches, the junction of bilateral mandibular processes, and apical ectodermal ridges of limb buds. At E11.5, apoptotic cells were noticeably decreased in most area, except the developing limbs and several somites in the tail region. In this study, the global temporospatial pattern of PCD throughout early development of mouse embryos was discussed. It may provide the basis for further studies on its role in the morphogenesis of the embryo.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6697-6701
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• 2013

Objective: To explore the inhibiting effect and mechanism of Endostar injection concomitant with cryoablation on lung adenocarcinoma A549 xenografts in nude mice. Materials and Methods: A total of 24 nude mice with subcutaneous xenografts of the A549 cell line were established and divided into 4 groups when the maximal diameters of tumors became 1 cm: control group, Endostar group, cryoablation group and combination group (Endostar concomitant with cryoablation). The nude mice were sacrificed after 21-days treatment, tumour tissues were removed to measure their volume, in situ test of TdT-mediated dUTP nick end labeling (TUNEL) was adopted to determine the cellular apoptosis around freezing injury zones, and immunohistochemical SP test was applied for the detection of micro-vessel density (MVD) and vascular endothelial growth factor (VEGF) expression levels. Results: At 21-days after treatment, the growth velocities of control group, Endostar group, cryoablation group and combination group were $236.7{\pm}51.2%$, $220.0{\pm}30.6%$, $159.5{\pm}29.3%$ and $103.3{\pm}25.5%$ (P<0.01), while cellular apoptosis rates of tumors were $21.7{\pm}2.34%$, ($22.17{\pm}1.47$)%, $38.3{\pm}1.37%$ and $49.2{\pm}1.72%$, (P<0.01), respectively, according to the immunohistochemical test. MVD and VEGF expression levels in the combination group were both lower than in other groups (P<0.01), also being positively related (r=0.925, P<0.01). Conclusions: Endostar can significantly improve the inhibitory effects of cryoablation on xenografts of lung adenocarcinoma A549, and the mechanism is probably associated with its function as an inhibitor of tumour neo-angiogenesis through down-regulating VEGF expression.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.5 no.1
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• pp.1-10
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• 2002

Purpose; Dysregulation of gastric epithelial cell proliferation and apoptosis are important in development of ulcer, atrophy and neoplasia in Helicobacter pylori (H. pylori) infection. The aim of this study was to investigate the effect of infection of H. pylori on gastric epithelial cell proliferation and apoptosis in children. Methods: Histological grading by updated Sydney system, PCNA immunostaining and TUNEL method were performed in H. pylori positive (N=58) and negative (N=40) gastric biopsy specimens. Results: In H. pylori positive children, there were significantly higher grade of polymorphonuclear neutrophil activity (P=0.000), chronic inflammation (P=0.000), epithelial damage (P=0.000) and lymphoid follicles (P=0.000) than in H. pylori negative children. Intestinal metaplasia was not seen in H. pylori positive children. PCNA index was significantly different between H. pylori positive children ($67.8{\pm}18.13$) and H. pylori negative children ($54.8{\pm}14.46$, P=0.000). There was positive correlation between PCNA index and H. pylori density (r=0.277, P=0.007), polymorphonuclear neutrophil activity (r=0.280, P=0.007) and chronic inflammation (r=0.284, P=0.006). Apoptosis index of H. pylori positive children ($0.44{\pm}0.447$) was significantly higher than of H. pylori negative children ($0.14{\pm}0.196$, P=0.000). There was positive correlation between apoptosis index and H. pylori density (r=0.472, P=0.000), polymorphonuclear neutrophil activity (r=0.370, P=0.001) and chronic inflammation (r=0.483, P=0.000). There was positive correlation between PCNA index and apoptosis index (r=0.353, P=0.003). Conclusion: The PCNA and apoptosis index in H. pylori positive children were significantly higher than in H. pylori negative children. This study suggested that gastric epithelial cell proliferation and apoptosis are important to pathogenesis of H. pylori infection in children.