scholarly journals DNA Fragmentation Follows Delayed Neuronal Death in CA1 Neurons Exposed to Transient Global Ischemia in the Rat

1997 ◽  
Vol 17 (9) ◽  
pp. 967-976 ◽  
Author(s):  
Carol K. Petito ◽  
Jorge Torres-Munoz ◽  
Brenda Roberts ◽  
John-Paul Olarte ◽  
Thaddeus S. Nowak ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Neuronal Death ◽  
Nuclear Dna ◽  
P53 Protein ◽  
Global Ischemia ◽  
Delayed Neuronal Death ◽  
Ca1 Neurons ◽  
Morphological Studies ◽  
Eosinophilic Cytoplasm

Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA precedes morphological changes in the nucleus and, later, in the cytoplasm. In ischemia, biochemical studies have detected oligonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than apoptosis. To resolve this apparent discrepancy, we subjected rats to 10 minutes of transient forebrain ischemia followed by 1 to 14 days of reperfusion. Parameters evaluated in the CA1 region of the hippocampus included morphology, in situ end labeling (ISEL) of fragmented DNA, and expression of p53. Neurons were indistinguishable from controls at postischemic day 1 but displayed cytoplasmic basophilia or focal condensations at day 2; some neurons were slightly swollen and a few appeared normal. In situ end labeling was absent. At days 3 and 5, approximately 40 to 60% of CA1 neurons had shrunken eosinophilic cytoplasm and pyknotic nuclei, but only half of these were ISEL. By day 14, many of the necrotic neurons had been removed by phagocytes; those remaining retained mild ISEL. Neither p53 protein nor mRNA were identified in control or postischemic brain by in situ hybridization with riboprobes or by northern blot analysis. These results show that DNA fragmentation occurs after the development of delayed neuronal death in CA1 neurons subjected to 10 minutes of global ischemia. They suggest that mechanisms other than apoptosis may mediate the irreversible changes in the CA1 neurons in this model.

Caffeine Releasable Stores of Ca2+ Show Depletion Prior to the Final Steps in Delayed CA1 Neuronal Death

2004 ◽  
Vol 92 (5) ◽  
pp. 2960-2967 ◽  
Author(s):  
Hong Xing ◽  
Aryan Azimi-Zonooz ◽  
C. William Shuttleworth ◽  
John A. Connor
Keyword(s):  
Endoplasmic Reticulum ◽  
Neuronal Death ◽  
Carotid Arteries ◽  
Cell Function ◽  
Mean Amplitude ◽  
Delayed Neuronal Death ◽  
Ischemic Insult ◽  
Cell Degeneration ◽  
Ca1 Neurons ◽  
The Mean

In addition to their role in signaling, Ca2+ ions in the endoplasmic reticulum also regulate important steps in protein processing and trafficking that are critical for normal cell function. Chronic depletion of Ca2+ in the endoplasmic reticulum has been shown to lead to cell degeneration and has been proposed as a mechanism underlying delayed neuronal death following ischemic insults to the CNS. Experiments here have assessed the relative content of ryanodine receptor-gated stores in CA1 neurons by measuring cytoplasmic Ca2+ increases induced by caffeine. These measurements were performed on CA1 neurons, in slice, from normal gerbils, and compared with responses from this same population of neurons 54–60 h after animals had undergone a standard ischemic insult: 5-min bilateral occlusion of the carotid arteries. The mean amplitude of responses in the postischemic population were less than one-third of those in control or sham-operated animals, and 35% of the neurons from postischemic animals showed very small responses that were ∼10% of the control population mean. Refilling of these stores after caffeine challenges was also impaired in postischemic neurons. These observations are consistent with our earlier finding that voltage-gated influx is sharply reduced in postischemic in CA1 neurons and the hypothesis that the resulting depletion in endosomal Ca2+ is an important cause of delayed neuronal death.

scholarly journals Genome-Wide Gene Expression Analysis for Induced Ischemic Tolerance and Delayed Neuronal Death following Transient Global Ischemia in Rats

2004 ◽  
Vol 24 (2) ◽  
pp. 212-233 ◽  
Author(s):  
Nobutaka Kawahara ◽  
Yan Wang ◽  
Akitake Mukasa ◽  
Kazuhide Furuya ◽  
Tatsuya Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Neuronal Death ◽  
Gene Expression Analysis ◽  
Ischemic Tolerance ◽  
Global Ischemia ◽  
Delayed Neuronal Death ◽  
Regulation Of Transcription ◽  
Genome Wide ◽  
A Genome

enome-wide gene expression analysis of the hippocampal CA1 region was conducted in a rat global ischemia model for delayed neuronal death and induced ischemic tolerance using an oligonucleotide-based DNA microarray containing 8,799 probes. The results showed that expression levels of 246 transcripts were increased and 213 were decreased following ischemia, corresponding to 5.1% of the represented probe sets. These changes were divided into seven expression clusters using hierarchical cluster analysis, each with distinct conditions and time-specific patterns. Ischemic tolerance was associated with transient up-regulation of transcription factors (c-Fos, JunB Egr-1, −2, −4, NGFI-B), Hsp70 and MAP kinase cascade-related genes (MKP-1), which are implicated cell survival. Delayed neuronal death exhibited complex long-lasting changes of expression, such as up-regulation of proapoptotic genes (GADD153, Smad2, Dral, Caspase-2 and −3) and down-regulation of genes implicated in survival signaling (MKK2, and PI4 kinase, DAG/PKC signaling pathways), suggesting an imbalance between death and survival signals. Our study provides a differential gene expression profile between delayed neuronal death and induced ischemic tolerance in a genome-wide analysis, and contributes to further understanding of the complex molecular pathophysiology in cerebral ischemia.

scholarly journals Possible Mechanism of Miltefosine-Mediated Death of Leishmania donovani

2004 ◽  
Vol 48 (8) ◽  
pp. 3010-3015 ◽  
Author(s):  
Navin K. Verma ◽  
Chinmoy S. Dey
Keyword(s):  
Dna Fragmentation ◽  
Mode Of Action ◽  
Leishmania Donovani ◽  
Nuclear Dna ◽  
Therapeutic Strategies ◽  
Dna Condensation ◽  
Intracellular Amastigotes ◽  
Labeling Method

ABSTRACT Miltefosine causes leishmanial death, but the possible mechanism(s) of action is not known. The mode of action of miltefosine was investigated in vitro in Leishmania donovani promastigotes as well as in extra- and intracellular amastigotes. Here, we demonstrate that miltefosine induces apoptosis-like death in L. donovani based on observed phenomena such as nuclear DNA condensation, DNA fragmentation with accompanying ladder formation, and in situ labeling of DNA fragments by the terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end labeling method. Understanding of miltefosine-mediated death will facilitate the design of new therapeutic strategies against Leishmania parasites.

scholarly journals Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.

1992 ◽  
Vol 119 (3) ◽  
pp. 493-501 ◽  
Author(s):  
Y Gavrieli ◽  
Y Sherman ◽  
S A Ben-Sasson
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Dna Fragmentation ◽  
Nuclear Dna ◽  
Nuclear Periphery ◽  
Lymphoid Tissues ◽  
Pooled Dna ◽  
Nuclear Dna Fragmentation ◽  
Small And Large Intestine

Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.

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