A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA.

Wijsman, Jan H.; Jonker, R. R.; Keijzer, Rob; Velde, C. J. H. van de; Cj, Cornelisse; Dierendonck, Jan Hein van

doi:10.1177/41.1.7678025

Cited by 776 publications

(421 citation statements)

References 9 publications

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“…The cerebrum was then sliced into 16 mm coronal brain sections. DNA fragmentation as a marker of overall cell death was ascertained by ISEL staining according to the protocol developed by Wijsman et al (1993). Sections were treated with Pronase E (1 g/mL; VWR International, Bridgeport, NJ, USA) and incubated with DNA polymerase I (50 mg/ mL; Fisher Scientific, Pittsburgh, PA, USA), biotin-21-2-deoxyuridine 5-triphosphate (10 mmol/L; BD Biosciences, San Jose, CA, USA), and deoxyadenosine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate (10 mmol/L; Fisher Scientific).…”

Section: Histologymentioning

confidence: 99%

“…Care was taken that ROIs were then positioned in the same location on H&E, ISEL as well as caspase-3-stained sections across animals. Lastly, since the ISEL procedure stains predominantly apoptotic (and to a lesser degree necrotic) cells (Wijsman et al, 1993), H&E staining was used to exemplarily confirm cell death. Briefly, neurons were classified as necrotic when they exhibited pyknosis, karyorrhexis, karyolysis, cytoplasmic eosinophilia ('red neuron'), or loss of affinity for hematoxylin ('ghost neuron') (Li et al, 2000).…”

Section: Histologymentioning

confidence: 99%

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Normobaric Hyperoxia Delays Perfusion/Diffusion Mismatch Evolution, Reduces Infarct Volume, and Differentially Affects Neuronal Cell Death Pathways after Suture Middle Cerebral Artery Occlusion in Rats

Henninger

Bouley

Nelligan

et al. 2007

J Cereb Blood Flow Metab

110

109

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Normobaric hyperoxia (NBO) has been shown to extend the reperfusion window after focal cerebral ischemia. Employing diffusion (DWI)-and perfusion (PWI)-weighted magnetic resonance imaging (MRI), the effect of NBO (100% started at 30 mins after middle cerebral artery occlusion (MCAO)) on the spatiotemporal evolution of ischemia during and after permanent (pMCAO) and transient suture middle cerebral artery occlusion (tMCAO) was investigated (experiment 3). In two additional experiments, time window (experiment 1) and cell death pathways (experiment 2) were investigated in the pMCAO model. In experiment 1, NBO treatment reduced infarct volume at 24 h after pMCAO by 10% when administered for 3 h (P > 0.05) and by 44% when administered for 6 h (P < 0.05). In experiment 2, NBO acutely (390 mins, P < 0.05) reduced in situ end labeling (ISEL) positivity in the ipsilesional penumbra but increased contralesional necrotic as well as caspase-3-mediated apoptotic cell death. In experiment 3, CBF characteristics and CBF-derived lesion volumes did not differ between treated and untreated animals, whereas the apparent diffusion coefficient (ADC)-derived lesion volume essentially stopped progressing during NBO treatment, resulting in a persistent PWI/DWI mismatch that could be salvaged by delayed (3 h) reperfusion. In conclusion, NBO (1) acutely preserved the perfusion/diffusion mismatch without altering CBF, (2) significantly extended the time window for reperfusion, (3) induced lasting neuroprotection in permanent ischemia, and (4) although capable of reducing cell death in hypoperfused tissue it also induced cell death in otherwise unaffected areas. Our data suggest that NBO may represent a promising strategy for acute stroke treatment.

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Section: Histologymentioning

confidence: 99%

Section: Histologymentioning

confidence: 99%

Normobaric Hyperoxia Delays Perfusion/Diffusion Mismatch Evolution, Reduces Infarct Volume, and Differentially Affects Neuronal Cell Death Pathways after Suture Middle Cerebral Artery Occlusion in Rats

Henninger

Bouley

Nelligan

et al. 2007

J Cereb Blood Flow Metab

110

109

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“…In situ end labelling The procedure used was based on that described by Wijsman et al [13]; formalin-fixed 4-m sections were cut from the paraffin wax tissue blocks, dewaxed, rehydrated and treated with 0 . 1% hydrogen peroxide for 15 min to block endogenous peroxidase activity.…”

Section: Routine Light Microscopymentioning

confidence: 99%

An analysis of apoptosis in lymphoid organs and lupus disease in murine systemic lupus erythematosus (SLE)

Ravirajan

Sarraf

Anilkumar

et al. 1996

Clinical and Experimental Immunology

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SUMMARYApoptosis is a programmed cell death process that helps to regulate both T cell and B cell development. In this study, we have investigated the levels of apoptotic death in cells of the thymuses and spleens (white matter) of autoimmune MRL-lpr/lpr mice with progressive lymphadenopathy and SLE disease activity; we also examined the renal pathology in these animals. Fas is a cell surface receptor, which when activated initiates the sequence of events that lead to apoptosis. In MRL-lpr/lpr mice Fas is defective, so the competency for apoptosis may be reduced. In young animals of advancing age the thymuses enlarged until in 5-month-old females the average weight was three times that at 1 month, and spleen and kidney weights also increased in size disproportionately. At light microscope level apoptotic cells in tissue sections were counted using both routine eosin and haematoxylin staining (to identify them by their morphology) and in situ end-labelling of cells with DNA strand breaks; their presence was further confirmed by electron microscopy. As the mice aged, the numbers of apoptotic cells in thymic cortex, thymic medulla and spleen white pulp areas reduced significantly (P < 0 . 01-0 . 001), whereas in BALB/c normal controls they increased significantly (P < 0 . 05). These changes were coincident with the development of severe lupus, whose activity was assessed by measuring serum anti-ssDNA and antidsDNA antibody titres and urinary protein (albumin) level which were elevated significantly by 5 months of age (P < 0 . 001 for both ssDNA and dsDNA and P < 0 . 01 for urine albumin) compared with their younger counterparts. Thus, lymphoid organ enlargement, decrease in apoptotic indices, elevated serum anti-ssDNA and anti-dsDNA antibody levels, and impaired renal function coincided with the onset and severity of lupus disease in lpr mice. It seems likely that there is a causal relationship between defective deletion of autoreactive lymphoid cells, imperfect Fas-mediated apoptosis and development of murine SLE.

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“…The method used to detect breaks in DNA strands in situ was adapted from those methods described by Gav rieli et al (1992) and Wijsman et al (1993). Following rehydration, tissue sections were permeabilized by incu bation in 0.25% pepsin (3,000 U/mg; Worthington Bio chemical Corporation, Freehold, NJ, U.S.A.) in diluted HCl (pH 2) for 30 min at 37°C, using 75 fJ.l per section.…”

Section: Detection Of Dna Breaks In Situ On Brain Sectionsmentioning

confidence: 99%

Differences in DNA Fragmentation following Transient Cerebral or Decapitation Ischemia in Rats

MacManus¹,

Hill²,

Preston³

et al. 1995

J Cereb Blood Flow Metab

113

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Summary:The time course of appearance of cells with DNA damage was studied in rats following transient se vere forebrain ischemia. This DNA damage could be de tected by in situ end-labeling on brain sections. The breaks in DNA appeared selectively by day I in the stri atum and later in the CAl region of the hippocampus. It was possible by double labeling to show that there was no DNA damage in astrocytes. The DNA breaks consisted of laddered DNA fragments indicative of an ordered ap optotic type of internucleosomal cleavage, which per sisted without smearing for up to 7 days of reperfusion. In

show abstract

A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA.

Cited by 776 publications

References 9 publications

Normobaric Hyperoxia Delays Perfusion/Diffusion Mismatch Evolution, Reduces Infarct Volume, and Differentially Affects Neuronal Cell Death Pathways after Suture Middle Cerebral Artery Occlusion in Rats

Normobaric Hyperoxia Delays Perfusion/Diffusion Mismatch Evolution, Reduces Infarct Volume, and Differentially Affects Neuronal Cell Death Pathways after Suture Middle Cerebral Artery Occlusion in Rats

An analysis of apoptosis in lymphoid organs and lupus disease in murine systemic lupus erythematosus (SLE)

Differences in DNA Fragmentation following Transient Cerebral or Decapitation Ischemia in Rats

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