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.

Extracellular ATP causes apoptosis and necrosis of cultured mesangial cells via P2Z/P2X7receptors

1998 ◽  
Vol 275 (6) ◽  
pp. F962-F971 ◽  
Author(s):  
Eckhard Schulze-Lohoff ◽  
Christian Hugo ◽  
Sylvia Rost ◽  
Susanne Arnold ◽  
Angela Gruber ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Mesangial Cells ◽  
Lucifer Yellow ◽  
Fold Increase ◽  
Extracellular Atp ◽  
Glomerular Disease ◽  
Terminal Deoxynucleotidyl Transferase ◽  
P53 Expression ◽  
Apoptosis And Necrosis

Mesangial cells undergo cell death both by apoptosis and necrosis during glomerular disease. Since nucleotides are released from injured and destroyed cells in the glomerulus, we examined whether extracellular ATP and its receptors may regulate cell death of cultured mesangial cells. Addition of extracellular ATP (300 μM to 5 mM) to cultured rat mesangial cells for 90 min caused a 5.8-fold increase in DNA fragmentation (terminal deoxynucleotidyl transferase assay) and a 4.2-fold increase in protein levels of the tumor suppressor p53, which is thought to regulate apoptosis. Apoptotic DNA fragmentation was confirmed by the diphenylamine assay and by staining with the DNA-specific fluorochrome Hoechst 33258. The necrotic markers, release of lactate dehydrogenase and uptake of trypan blue, were not positive before 3 h of ATP addition. The effects of ATP on DNA fragmentation and p53 expression were reproduced by the purinergic P2Z/P2X7 receptor agonist, 3′- O-(4-benzoylbenzoyl)-ATP, and inhibited by the P2Z/P2X7 receptor blocker, oxidized ATP. Transcripts encoding the P2Z/P2X7 receptor were expressed by cultured mesangial cells as determined by Northern blot analysis. P2Z/P2X7 receptor-associated pore formation in the plasma membrane was demonstrated by the Lucifer yellow assay. We conclude that activation of P2Z/P2X7 receptors by extracellular ATP causes apoptosis and necrosis of cultured mesangial cells. Activation of purinergic P2Z/P2X7 receptors may play a role in causing death of mesangial cells during glomerular disease.

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.

Chromatin supraorganization, DNA fragmentation, and cell death in snake erythrocytes

2005 ◽  
Vol 83 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Maristela Miyamoto ◽  
Benedicto C Vidal ◽  
Maria Luiza S Mello
Keyword(s):  
Cell Death ◽  
Single Cell ◽  
Acid Hydrolysis ◽  
Dna Fragmentation ◽  
Gel Electrophoresis ◽  
Cell Biology ◽  
Tunel Assay ◽  
Snake Species ◽  
Single Cell Gel Electrophoresis ◽  
Bothrops Alternatus

In nucleate erythrocytes of several vertebrate groups, the frequency and intensity of DNA fragmentation associated with programed cell death vary considerably. Although hemoglobin efficiency may be related to erythrocyte life span, and hemoglobin types and erythrocyte life spans are assumed to vary in reptiles, no data on DNA fragmentation and chromatin organization as related to cell death exist for snakes. In the present study, chromatin supraorganization, DNA fragmentation, and cell death were investigated in four snake species (Crotalus durissus terrificus, Bothrops jararaca, Bothrops alternatus, and Bothrops neuwiedii), which differ in their geographical distribution and habitats, by using image analysis of Feulgen hydrolysis kinetics, the TUNEL assay, single-cell gel electrophoresis, and transmission electron microscopy. Relatively few circulating erythrocytes were found to be simultaneously committed to cell death, although there was some variation among the snake species. Conspicuous nuclear and cytoplasmic organelles suggestive of metabolic activity were seen ultrastructurally in most snake erythrocytes. The DNA of the snake erythrocyte chromatin was much more resistant to Feulgen acid hydrolysis (DNA depurination and breakdown) than that of young adult bullfrog erythrocytes, which had a high frequency and intensity of DNA fragmentation. Of the species studied, B. neuwiedii and C. d. terrificus showed the greatest resistance to Feulgen acid hydrolysis and to the DNA fragmentation, revealed by the TUNEL assay. Although B. neuwiedii also showed the lowest frequency of cells with more damaged DNA in the single-cell gel electrophoresis assay, C. d. terrificus had the highest frequency of damaged cells, possibly because of the abundance of alkaline-sensitive DNA sites. The results for DNA fragmentation and cell death in erythrocytes of B. jararaca and B. alternatus generally differed from those for C. d. terrificus and B. neuwiedii and may reflect differences in the biology of these species selected under different geographical habitats. The differences in erythrocyte cell biology reported here may be related to hemoglobin variants selected in the mentioned snake species and that would lead the cells to different resistances to unfavorable environmental conditions.Key words: erythrocytes, snakes, DNA fragmentation, chromatin supraorganization, cell death.

scholarly journals Inhibition of Interleukin-1β Converting Enzyme Family Proteases (Caspases) Reduces Cold Injury-Induced Brain Trauma and DNA Fragmentation in Mice

1999 ◽  
Vol 19 (6) ◽  
pp. 634-642 ◽  
Author(s):  
Yuiko Morita-Fujimura ◽  
Miki Fujimura ◽  
Makoto Kawase ◽  
Kensuke Murakami ◽  
Gyung Whan Kim ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Brain Trauma ◽  
Cold Injury ◽  
Interleukin 1Β ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Converting Enzyme ◽  
Caspase Family

The authors examined the effect of z-VAD.FMK, an inhibitor that blocks caspase family proteases, on cold injury-induced brain trauma, in which apoptosis as well as necrosis is assumed to play a role. A vehicle alone or with z-VAD.FMK was administered into the cerebral ventricles of mice 15 minutes before and 24 and 48 hours after cold injury. At 24 hours after cold injury, infarction volumes in the z-VAD.FMK-treated animals were significantly smaller than infarction volumes in the vehicle-treated animals, and were further decreased at 72 hours (0.92 ± 1.80 mm3, z-VAD.FMK-treated animals; 7.46 ± 3.53 mm3, vehicle-treated animals; mean ± SD, n = 7 to 8). The amount of DNA fragmentation was significantly decreased in the z-VAD.FMK-treated animals compared with the vehicle-treated animals, as shown by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling staining and DNA gel electrophoresis. By Western blot analysis, both the proform and activated form of interleukin-1β converting enzyme (caspase 1) were detected in the control brain, and the activated form showed moderate reduction after cold injury-induced brain trauma. These results indicate that caspase inhibitors could reduce cold injury-induced brain trauma by preventing neuronal cell death by DNA damage. The caspase family proteases appear to contribute to the mechanisms of cell death in cold injury-induced brain trauma and to provide therapeutic targets for traumatic brain injury.

scholarly journals In situ DNA fragmentation during the re-establishment of desiccation tolerance in germinated seeds of Cedrela fissilis Vell.

2019 ◽  
Vol 41 (2) ◽  
pp. 244-249
Author(s):  
Tathiana Elisa Masetto ◽  
José Marcio Rocha Faria
Keyword(s):  
Cell Death ◽  
Moisture Content ◽  
Dna Fragmentation ◽  
Desiccation Tolerance ◽  
Cell Injury ◽  
Tunel Staining ◽  
Long Term Storage ◽  
Germinated Seeds

Abstract: Dehydration is a necessary procedure prior to exposing seeds to long term storage, but this is associated with metabolism-linked injury mediated by cell injury. In order to assess cellular alterations during re-establishment of desiccation tolerance (DT) in C. fissilis germinated seeds and their relation to DNA damage, we verified the occurrence of DNA fragmentation through the TUNEL test and its evidence through the cytological analyses. To re-establish DT, germinated seeds were incubated for 72 h in polyethylene glycol (PEG, -2.04 MPa) before dehydration in silica gel (at 10% moisture content) followed by rehydration. The moisture content changes during the reestablishment of the desiccation tolerance was accomplished. (DT)TdT-dUPT terminal nick-end labeling (TUNEL) was used to assess rates of cell death. TUNEL staining was performed using Click-iT-TUNEL Alexa Flour imaging assay. The TUNEL test showed a consistent DNA fragmentation in the 2 and 5 mm long radicles. Moreover, nuclear and chromosomal alterations were observed in the 5 mm meristematic root cell cycle, contributing to the identification of diagnostic markers of cell death.

scholarly journals Inflammation and kidney injury attenuated by prior intake of Brazil nuts in the process of ischemia and reperfusion

2018 ◽  
Vol 40 (4) ◽  
pp. 312-318
Author(s):  
Maria Fernanda Ribeiro Cury ◽  
Estéfany Queiroz Olivares ◽  
Renata Correia Garcias ◽  
Giovana Queda Toledo ◽  
Natassia Alberici Anselmo ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Cell Injury ◽  
Kidney Injury ◽  
Left Renal Artery ◽  
Ischemia And Reperfusion ◽  
Brazil Nuts ◽  
Markers Of Inflammation ◽  
Cox 2 ◽  
Renal Expression

ABSTRACT Introduction: Ischemia and reperfusion (IR) is a process inherent to the procedures involved in the transplantation of organs that causes inflammation, cell death and cell injury, and may lead to rejection of the graft. It is possible that the anti-inflammatory properties of the Brazil nuts (BN) can mitigate the renal injury caused by IR. Objective: To investigate whether the previous intake of BN reduces the expression of markers of inflammation, injury, and cell death after renal IR. Methods: Male Wistar rats were distributed into six groups (N = 6/group): SHAM (control), SHAM treated with 75 or 150 mg of BN, IR, and IR treated with 75 or 150 mg of BN. The IR procedure consisted of right nephrectomy and occlusion of the left renal artery with a non-traumatic vascular clamp for 30 min. BN was given daily from day 1 to 7 before surgery (SHAM or IR), and maintained until sacrifice (48 h after surgery). Inflammation was evaluated by renal expression of COX-2 and TGF-β, injury by the expression of vimentin, and cell death by apoptosis through caspase-3 expression (immunohistochemistry). Results: Pretreatment with 75 mg of BN reduced renal expression of the COX-2, TGF-β, vimentin, and caspase-3. The dose of 150 mg caused increased expression of COX-2. Conclusion: In experimental IR, the damage can be minimized with a prior low-dose intake of BN, improving inflammation, injury, and cell death.

In situ end labeling of fragmented DNA in induced ovarian atresia

1994 ◽  
Vol 72 (11-12) ◽  
pp. 573-579 ◽  
Author(s):  
Katharina D'herde ◽  
Guido De Pestel ◽  
Frank Roels
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Granulosa Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dna Fragments ◽  
Dna Polymerase I ◽  
Late Event ◽  
Chromatin Configuration ◽  
Polymerase I

Apoptosis is studied in a model of induced follicular atresia in the ovary of Japanese quail (Coturnix coturnix japonica) by in situ end labeling of DNA fragments in granulosa cells using two different techniques (incorporation of labeled nucleotides by DNA polymerase I or terminal deoxynucleotidyl transferase). The most remarkable observation related to apoptosis in this model is the predominant cytoplasmic localization of labeled DNA fragments, while DNA fragmentation appears to be absent from compacted chromatin masses of apoptotic nuclei and apoptotic nuclear fragments. Unstained apoptotic bodies are present adjacent to stained ones, so that their detection rate on hematoxylin + eosin stained sections is better than on the in situ end-labeled sections. This suggests that DNA fragmentation is a late event or not obligatory in apoptotic granulosa cell death. In contrast to similar studies on atretic granulosa in mammalian models, the process of apoptosis is asynchronous in the granulosal epithelium, with a majority of nuclei with normal chromatin configuration remaining negative for DNA fragmentation. Finally it is shown that the techniques used are not specific for apoptosis, as DNA fragmentation in necrotic granulosa cells is detected as well.Key words: programmed cell death, apoptosis, in situ DNA end labeling, endonuclease, necrosis.

scholarly journals Acute Plasmalemma Permeability and Protracted Clearance of Injured Cells after Controlled Cortical Impact in Mice

2007 ◽  
Vol 28 (3) ◽  
pp. 490-505 ◽  
Author(s):  
Michael J Whalen ◽  
Turgay Dalkara ◽  
Zerong You ◽  
Jianhua Qiu ◽  
Daniela Bermpohl ◽  
...  
Keyword(s):  
Cell Death ◽  
Propidium Iodide ◽  
Time Course ◽  
Injury Severity ◽  
Cell Injury ◽  
Membrane Damage ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cellular Injury ◽  
Controlled Cortical Impact ◽  
Cortical Impact

Cell death after traumatic brain injury (TBI) evolves over days to weeks. Despite advances in understanding biochemical mechanisms that contribute to posttraumatic brain cell death, the time course of cell injury, death, and removal remains incompletely characterized in experimental TBI models. In a mouse controlled cortical impact (CCI) model, plasmalemma permeability to propidium iodide (PI) was an early and persistent feature of posttraumatic cellular injury in cortex and hippocampus. In cortical and hippocampal brain regions known to be vulnerable to traumatic cell death, the number of PI + cells peaked early after CCI, and increased with increasing injury severity in hippocampus but not cortex ( P < 0.05). Propidium iodide labeling correlated strongly with hematoxylin and eosin staining in injured cells ( r = 0.99, P < 0.001), suggesting that plasmalemma damage portends fatal cellular injury. Using PI pulse labeling to identify and follow the fate of a cohort of injured cells, we found that many PI+ cells recovered plasmalemma integrity by 24 h and were terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling negative, but nonetheless disappeared from injured brain by 7 days. Propidium iodide-positive cells in dentate gyrus showed significant ultrastructural damage, including plasmalemma and nuclear membrane damage or overt membrane loss, in all cells when examined by laser capture microdissection and transmission electron microscopy 1 to 24 h after CCI. The data suggest that plasmalemma damage is a fundamental marker of cellular injury after CCI; some injured cells might have an extended window for potential rescue by neuroprotective strategies.

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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