scholarly journals Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick End Labeling (TUNEL) Assay to Characterize Histopathologic Changes Following Thermal Injury

2018 ◽  
Vol 30 (1) ◽  
pp. 41 ◽  
Author(s):  
Ji Min Lee ◽  
Ji Hyun Park ◽  
Bo Young Kim ◽  
Il-Hwan Kim
Keyword(s):  
Thermal Injury ◽  
Tunel Assay ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Deoxyuridine Triphosphate ◽  
Histopathologic Changes

scholarly journals TUNEL Assay: A Powerful Tool for Kidney Injury Evaluation

2021 ◽  
Vol 22 (1) ◽  
pp. 412
Author(s):  
Christopher L. Moore ◽  
Alena V. Savenka ◽  
Alexei G. Basnakian
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Cell Injury ◽  
Kidney Injury ◽  
Cell Types ◽  
Tunel Assay ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Hypoxic Injury ◽  
Additional Information ◽  
Tissue Compartments

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay is a long-established assay used to detect cell death-associated DNA fragmentation (3’-OH DNA termini) by endonucleases. Because these enzymes are particularly active in the kidney, TUNEL is widely used to identify and quantify DNA fragmentation and cell death in cultured kidney cells and animal and human kidneys resulting from toxic or hypoxic injury. The early characterization of TUNEL as an apoptotic assay has led to numerous misinterpretations of the mechanisms of kidney cell injury. Nevertheless, TUNEL is becoming increasingly popular for kidney injury assessment because it can be used universally in cultured and tissue cells and for all mechanisms of cell death. Furthermore, it is sensitive, accurate, quantitative, easily linked to particular cells or tissue compartments, and can be combined with immunohistochemistry to allow reliable identification of cell types or likely mechanisms of cell death. Traditionally, TUNEL analysis has been limited to the presence or absence of a TUNEL signal. However, additional information on the mechanism of cell death can be obtained from the analysis of TUNEL patterns.

scholarly journals Melatonin and/or erythropoietin combined with hypothermia in a piglet model of perinatal asphyxia

2020 ◽  
Author(s):  
Raymand Pang ◽  
Adnan Avdic-Belltheus ◽  
Christopher Meehan ◽  
Kathryn Martinello ◽  
Tatenda Mutshiya ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Triple Therapy ◽  
Sensorimotor Cortex ◽  
Perinatal Asphyxia ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Resonance Spectroscopy ◽  
Periventricular White Matter ◽  
Deoxyuridine Triphosphate ◽  
Hypoxia Ischemia ◽  
Effective Adjunct

Abstract As therapeutic hypothermia is only partially protective for neonatal encephalopathy, safe and effective adjunct therapies are urgently needed. Melatonin and erythropoietin show promise as safe and effective neuroprotective therapies. We hypothesized that melatonin and erythropoietin individually augment 12-hour hypothermia (double therapies) and hypothermia + melatonin + erythropoietin (triple therapy) leads to optimal brain protection. Following carotid artery occlusion and hypoxia, 49 male piglets (<48 hours old) were randomized to: (i) hypothermia + vehicle (n = 12), (ii) hypothermia + melatonin (20 mg/kg over 2 hours) (n = 12), (iii) hypothermia + erythropoietin (3000 U/kg bolus) (n = 13) or (iv) triple therapy (n = 12). Melatonin, erythropoietin or vehicle were given at 1, 24 and 48 hours after hypoxia-ischemia. Hypoxia-ischemia severity was similar across groups. Therapeutic levels were achieved 3 hours after hypoxia-ischemia for melatonin (15-30mg/L) and within 30 minutes of erythropoietin administration (maximum concentration 10,000 mU/mL). Compared to hypothermia + vehicle, we observed faster amplitude integrated EEG recovery from 25-30 hours with hypothermia + melatonin (p = 0.02) and hypothermia + erythropoietin (p = 0.033) and from 55-60 hours with triple therapy (p = 0.042). Magnetic Resonance Spectroscopy Lactate/N-acetyl aspartate peak ratio was lower at 66 hours in hypothermia + melatonin (p = 0.012) and triple therapy (p = 0.032). With hypothermia + melatonin, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelled-positive cells were reduced in sensorimotor cortex (p = 0.017) and oligodendrocyte transcription factor 2 labelled-positive counts increased in hippocampus (p = 0.014) and periventricular white matter (p = 0.039). There was no reduction in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelled-positive cells with hypothermia + erythropoietin, but increased oligodendrocyte transcription factor 2 labelled-positive cells in 5 of 8 brain regions (p < 0.05). Overall, melatonin and erythropoietin were safe and effective adjunct therapies to hypothermia. Hypothermia + melatonin double therapy led to faster amplitude integrated EEG recovery, amelioration of Lactate/N-acetyl aspartate rise and reduction in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelled-positive cells in the sensorimotor cortex. Hypothermia + erythropoietin double therapy was association with EEG recovery and was most effective in promoting oligodendrocyte survival. Triple therapy provided no added benefit over the double therapies in this 72-hour study. Melatonin and erythropoietin influenced cell death and oligodendrocyte survival differently, reflecting distinct neuroprotective mechanisms which may become more visible with longer term studies. Staggering the administration of therapies with early melatonin and later erythropoietin (after hypothermia) may provide better protection; each therapy has complementary actions which may be time critical during the neurotoxic cascade after hypoxia-ischemia.

Induction of apoptosis in indole-3-carbinol-treated lung cancer H1299 cells via ROS level elevation

2020 ◽  
pp. 096032712096996
Author(s):  
Heui Min Lim ◽  
See-Hyoung Park ◽  
Myeong Jin Nam
Keyword(s):  
Lung Cancer ◽  
Tunel Assay ◽  
Human Lung Cancer ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Ros Generation ◽  
Dependent Manner ◽  
Detection Assay ◽  
Caspase 7 ◽  
Level Elevation ◽  
Apoptotic Proteins

This study was focused on investigating the anticancer potential of indole-3-carbinol (I3C) against lung cancer H1299 cells via an increase in ROS levels. To investigate the induction of growth arrest and/or cell death in H1299 cells, a cell cycle arrest assay, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) assay, and reactive oxygen species (ROS) detection assay were performed. Through the TUNEL assay, we detected I3C-induced DNA fragmentation. Fluorescence-activated cell sorting (FACS) analysis showed that I3C induced an increase in ROS levels and apoptotic rate in a dose- and time-dependent manner in H1299 cells. Western blotting demonstrated that activated forms of caspase-3, caspase-7, caspase-9, and poly (ADP-ribose) polymerase (PARP) were increased in I3C-treated H1299 cells following treatment with I3C. Furthermore, protein expression levels of FOXO3, bim, bax, and phosphorylated ERK and JNK were increased, while those of pAkt, Bcl-xL, and Bcl-2 were decreased by I3C treatment of H1299 cells. To confirm the relationship between cell apoptosis and ROS generation, H1299 cells were treated with I3C simultaneously with N-acetylcysteine (NAC), and it was shown that ROS levels decreased and viability increased. Moreover, in western blot analysis, expression of anti-apoptotic proteins (thioredoxin1, peroxiredoxin-1, Bcl-2, and Bcl-xL) in I3C-treated cells was evidently downregulated and pro-apoptotic proteins (active ASK1 and cleaved PARP) were upregulated compared to cells co-treated with NAC. The study showed that I3C induced downregulation of ROS regulator proteins and elevation of ROS, thus activating apoptotic signaling cascades in human lung cancer H1299 cells.

scholarly journals Comparison of Proton and Photon Beam Irradiation in Radiation-Induced Intestinal Injury Using a Mouse Model

2019 ◽  
Vol 20 (8) ◽  
pp. 1894 ◽  
Author(s):  
Changhoon Choi ◽  
Chansu Lee ◽  
Sung-Won Shin ◽  
Shin-Yeong Kim ◽  
Sung Noh Hong ◽  
...  
Keyword(s):  
Proton Beam ◽  
Mouse Models ◽  
Proton Irradiation ◽  
Apoptotic Cell ◽  
Proton Beam Therapy ◽  
Tunel Assay ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Limiting Factor ◽  
X Rays ◽  
X Ray

When radiotherapy is applied to the abdomen or pelvis, normal tissue toxicity in the gastrointestinal (GI) tract is considered a major dose-limiting factor. Proton beam therapy has a specific advantage in terms of reduced doses to normal tissues. This study investigated the fundamental differences between proton- and X-ray-induced intestinal injuries in mouse models. C57BL/6J mice were irradiated with 6-MV X-rays or 230-MeV protons and were sacrificed after 84 h. The number of surviving crypts per circumference of the jejunum was identified using Hematoxylin and Eosin staining. Diverse intestinal stem cell (ISC) populations and apoptotic cells were analyzed using immunohistochemistry (IHC) and a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay, respectively. The crypt microcolony assay revealed a radiation-dose-dependent decrease in the number of regenerative crypts in the mouse jejunum; proton irradiation was more effective than X-ray irradiation with a relative biological effectiveness of 1.14. The jejunum is the most sensitive to radiations, followed by the ileum and the colon. Both types of radiation therapy decreased the number of radiosensitive, active cycling ISC populations. However, a higher number of radioresistant, reserve ISC populations and Paneth cells were eradicated by proton irradiation than X-ray irradiation, as shown in the IHC analyses. The TUNEL assay revealed that proton irradiation was more effective in enhancing apoptotic cell death than X-ray irradiation. This study conducted a detailed analysis on the effects of proton irradiation versus X-ray irradiation on intestinal crypt regeneration in mouse models. Our findings revealed that proton irradiation has a direct effect on ISC populations, which may result in an increase in the risk of GI toxicity during proton beam therapy.

Evaluation of Sperm DNA Fragmentation via Halosperm Technique and TUNEL Assay Before and After Cryopreservation

2019 ◽  
Vol 26 (12) ◽  
pp. 1575-1581 ◽  
Author(s):  
Senay Cankut ◽  
Turgay Dinc ◽  
Mehmet Cincik ◽  
Guler Ozturk ◽  
Belgin Selam
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Tunel Assay ◽  
University Hospital ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sperm Dna Fragmentation ◽  
Liquid Form ◽  
Sperm Dna Damage ◽  
Sperm Dna ◽  
Before And After

Aim: Human sperm DNA fragmentation is one of the factors suggested for male infertility. The ratio of sperm DNA damage in semen may adversely affect both the fertilization rate and the embryo development of in vitro fertilization/ intracytoplasmic sperm injection cycles. Sperm cryopreservation both increases the success rates in assisted reproductive techniques (ARTs) and contributes to the preservation of fertility before testis surgery, chemotherapy, and radiotherapy. The aim of the current study is to determine sperm DNA fragmentation, following cryopreservation. Methods: A cross-sectional, observational study was conducted at a university hospital infertility clinic. One hundred (n = 100) volunteer fertile men (ages between 21 and 39 years) with normozoospermic sperm parameters were involved in the current study. Sperm DNA damage was evaluated with the Halosperm technique and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Fresh samples were studied in liquid form. The remaining samples were kept frozen and then thawed after 1 month and reevaluated with the Halosperm technique and TUNEL assay. Results were then compared between the fresh and frozen samples. Results: Sperm DNA fragmentation results with the Halosperm technique both before and after cryopreservation were 25% (5%-65%) and 40% (6%-89%), respectively, with a statistically significant increase (15%; P < .001). Sperm DNA fragmentation results by TUNEL assay before and after cryopreservation were 17% (3%-43%) and 36% (7%-94%), respectively, with a statistically significant increase (19%; P <.001). Conclusion: The current data demonstrate increased sperm DNA damage after cryopreservation. Further studies may contribute to development of less harmful techniques and cryoprotectants in order to improve the results of ART.

Zinc and selenium combination treatment protected diabetes-induced testicular and epididymal damage in rat

2020 ◽  
Vol 39 (9) ◽  
pp. 1235-1256 ◽  
Author(s):  
C Sahu ◽  
DK Dwivedi ◽  
GB Jena
Keyword(s):  
Glutathione Peroxidase ◽  
Germ Cell ◽  
Combination Treatment ◽  
Cell Damage ◽  
Combined Treatment ◽  
Tunel Assay ◽  
The Body ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Feed Water ◽  
Related Factor

Diabetes increases the possibility of germ cell damage, hypogonadism, and male infertility. Diabetic condition negatively impacts zinc (Zn) and selenium (Se) levels in the body. Zn and Se are among the most important trace elements involved in the regulation of redox reaction, antioxidants enzymes activities, and DNA expression in a germ cell. The present study aimed to elucidate the combined effects of Zn and Se treatment on diabetes-induced germ cell damage in male Sprague Dawley rats. Type 1 diabetes was induced by the single intraperitoneal (i.p.) injection of streptozotocin (55 mg/kg). Zn (3 mg/kg, i.p.) and Se (0.5 mg/kg, i.p.) were administered daily for 8 consecutive weeks. All the animals were provided with normal feed and water throughout the study. The effects on germ cell damage were evaluated by body weight, feed-water intake, organ weight, sperm count, motility, sperm head morphology, biochemical analysis, histology, immunohistochemistry, halo assay, germ cell comet assay, testes terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling (TUNEL) assay, sperm TUNEL assay, serum protein pattern analysis, and subcellular analysis using transmission electron microscopy. Further, the expressions of nuclear erythroid-derived related factor 2, catalase, glutathione peroxidase 4, and glutathione peroxidase 5 were carried out to ascertain the mechanism of protection. The present results demonstrated that 8 weeks combined treatment of Zn (3 mg/kg, i.p.) and Se (0.5 mg/kg, i.p.) reduced diabetes-induced germ cell damage. This study further highlighted that Zn and Se combination treatment might be a better strategy for the germ cell protection in diabetes and deserve further investigation.

Histopathologic Changes After Bipolar Resection of the Prostate: Depth of Penetration of Bipolar Thermal Injury

2012 ◽  
Vol 26 (10) ◽  
pp. 1367-1371 ◽  
Author(s):  
Michael Maddox ◽  
Gyan Pareek ◽  
Shadi Al Ekish ◽  
Simone Thavaseelan ◽  
Akanksha Mehta ◽  
...  
Keyword(s):  
Thermal Injury ◽  
Depth Of Penetration ◽  
Histopathologic Changes

scholarly journals Down-regulation of MicroRNA-21 Is Involved in the Propofol-induced Neurotoxicity Observed in Human Stem Cell–derived Neurons

2014 ◽  
Vol 121 (4) ◽  
pp. 786-800 ◽  
Author(s):  
Danielle M. Twaroski ◽  
Yasheng Yan ◽  
Jessica M. Olson ◽  
Zeljko J. Bosnjak ◽  
Xiaowen Bai
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Signal Transducer ◽  
Deoxyuridine Triphosphate ◽  
Transcription 3

Abstract Background: Recent studies in various animal models have suggested that anesthetics such as propofol, when administered early in life, can lead to neurotoxicity. These studies have raised significant safety concerns regarding the use of anesthetics in the pediatric population and highlight the need for a better model to study anesthetic-induced neurotoxicity in humans. Human embryonic stem cells are capable of differentiating into any cell type and represent a promising model to study mechanisms governing anesthetic-induced neurotoxicity. Methods: Cell death in human embryonic stem cell–derived neurons was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling staining, and microRNA expression was assessed using quantitative reverse transcription polymerase chain reaction. miR-21 was overexpressed and knocked down using an miR-21 mimic and antagomir, respectively. Sprouty 2 was knocked down using a small interfering RNA, and the expression of the miR-21 targets of interest was assessed by Western blot. Results: Propofol dose and exposure time dependently induced significant cell death (n = 3) in the neurons and down-regulated several microRNAs, including miR-21. Overexpression of miR-21 and knockdown of Sprouty 2 attenuated the increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling–positive cells following propofol exposure. In addition, miR-21 knockdown increased the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling–positive cells by 30% (n = 5). Finally, activated signal transducer and activator of transcription 3 and protein kinase B (Akt) were down-regulated, and Sprouty 2 was up-regulated following propofol exposure (n = 3). Conclusions: These data suggest that (1) human embryonic stem cell–derived neurons represent a promising in vitro human model for studying anesthetic-induced neurotoxicity, (2) propofol induces cell death in human embryonic stem cell–derived neurons, and (3) the propofol-induced cell death may occur via a signal transducer and activator of transcription 3/miR-21/Sprouty 2–dependent mechanism.

Sequential and spatial profiles of apoptosis in ischemic penumbra after two-vein occlusion in rats

2006 ◽  
Vol 104 (6) ◽  
pp. 938-944 ◽  
Author(s):  
Toshikazu Nishioka ◽  
Hiroyuki Nakase ◽  
Mitsutoshi Nakamura ◽  
Noboru Konishi ◽  
Toshisuke Sakaki
Keyword(s):  
Immunohistochemical Analysis ◽  
Tunel Assay ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Therapeutic Window ◽  
Ischemic Penumbra ◽  
The Core ◽  
Bax Protein ◽  
Vein Occlusion ◽  
The Relationship

Object The two-vein occlusion model is known to be useful for ischemic penumbra studies in vivo. It was applied here to examine sequential changes in the expression of Bax and Bcl-2 proteins and in apoptotic cells to assess the relationship between penumbra and apoptosis. Methods Two cortical veins were occluded photochemically by using rose bengal dye in 27 Wistar rats. The animals were killed with perfusion fixation at the following intervals: 4, 12, 24, 48, 96, and 168 hours after vein occlusion (four at each interval; three additional rats were sham-treated). Immunohistochemical analysis for the Bcl-2 family of proteins was performed along with the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay to examine the relationship to single-cell death. Cells positive for antiapoptotic proteins began to appear in the TUNEL assay for animals killed 24 hours after vein occlusion, with a peak at 48 hours. These cells were localized in the core of infarction. Immunohistochemical staining for Bax protein showed an increased presence around ischemic lesions at 4 hours after vein occlusion, and the amounts continued to rise until 24 hours, when the localization was diffuse around the core of infarction. Negative findings on immunohistochemical studies for Bcl-2 protein were seen at the early phase after two-vein occlusion. Conclusions After vein occlusion, apoptosis appeared sequentially and widely in cortical lesions considered to be the penumbra. Therefore, control of apoptosis would be expected to offer a therapeutic window for treatment of venous infarction.

scholarly journals Do TUNEL and Other Apoptosis Assays Detect Cell Death in Preclinical Studies?

2020 ◽  
Vol 21 (23) ◽  
pp. 9090
Author(s):  
Razmik Mirzayans ◽  
David Murray
Keyword(s):  
Cell Death ◽  
Genomic Dna ◽  
Apoptotic Cell ◽  
Tunel Assay ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Caspase Activation ◽  
Dna Breaks ◽  
Tissue Samples ◽  
Dna Breakage

The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay detects DNA breakage by labeling the free 3ʹ-hydroxyl termini. Given that genomic DNA breaks arise during early and late stages of apoptosis, TUNEL staining continues to be widely used as a measure of apoptotic cell death. The advantages of the assay include its relative ease of performance and the broad availability of TUNEL assay kits for various applications, such as single-cell analysis of apoptosis in cell cultures and tissue samples. However, as briefly discussed herein, aside from some concerns relating to the specificity of the TUNEL assay itself, it was demonstrated some twenty years ago that the early stages of apoptosis, detected by TUNEL, can be reversed. More recently, compelling evidence from different biological systems has revealed that cells can recover from even late stage apoptosis through a process called anastasis. Specifically, such recovery has been observed in cells exhibiting caspase activation, genomic DNA breakage, phosphatidylserine externalization, and formation of apoptotic bodies. Furthermore, there is solid evidence demonstrating that apoptotic cells can promote neighboring tumor cell repopulation (e.g., through caspase-3-mediated secretion of prostaglandin E2) and confer resistance to anticancer therapy. Accordingly, caution should be exercised in the interpretation of results obtained by the TUNEL and other apoptosis assays (e.g., caspase activation) in terms of apoptotic cell demise.

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