(Stroke. 1995;26:1252-1258.)
© 1995 American Heart Association, Inc.
Articles |
Induction of DNA Fragmentation After 10 to 120 Minutes of Focal Cerebral Ischemia in Rats
From the Department of Neurology, Henry Ford Health Science Center, Detroit (Y.L., M.C., N.J., Z.G.Z., C.Z.), and the Department of Physics, Oakland University, Rochester (M.C., Z.G.Z.), Mich.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Background and Purpose The induction of neuronal necrosis has been studied after various durations of transient middle cerebral artery (MCA) occlusion in the rat. The objective of the present study was to measure the numbers and anatomic distribution of cells exhibiting apoptotic bodies as an indication of DNA fragmentation and apoptotic cell death as a function of duration of transient MCA occlusion in the rat.
Methods The MCA of male Wistar rats (n=24) was occluded for 10, 20, 30, 60, 90, and 120 minutes (n=4 per group) with the use of an intraluminal monofilament, and reperfusion was instituted for 48 hours. DNA fragmentation was measured in paraffin sections with the use of a terminal deoxynucleotidyl- transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method. Adjacent sections were stained with hematoxylin and eosin for analysis of ischemic cell damage, and immunohistochemical double staining methods were used for cell identification. Sham-operated rats (n=4) and normal rats not subjected to any surgical procedure (n=4) were used as controls for apoptosis detection.
Results Within 5-µm-thick coronal sections, DNA fragmentation was present in 0 to 3 apoptotic cells in each hemisphere of normal, sham-operated rats as well as in the contralateral hemisphere of ischemic rats. After 10 to 20 minutes of MCA occlusion, apoptotic cells exhibiting DNA fragmentation (10 to 20) increased in the regions of selective neuronal necrosis in the preoptic area and in the striatum. After 30 to 60 minutes of ischemia, scattered apoptotic cells (30 to 60) exhibited DNA fragmentation and expanded into areas of selective neuronal necrosis in the cortex. After 90 to 120 minutes of occlusion, groups of apoptotic cells (70 to 200, >95% neurons) were primarily localized to the inner boundary zone of the infarct.
Conclusions A range of mild to severe ischemia-reperfusion stimuli induce internucleosomal DNA cleavage. The presence and anatomic location of apoptotic cells exhibiting DNA fragmentation after transient cerebral occlusion indicate that apoptosis accompanies neuronal necrosis.
Key Words: apoptosis • DNA • middle cerebral artery • occlusion • rats
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Apoptosis describes a specific form of cell death that has been characterized morphologically and biochemically.1 2 Apoptosis is an essential strategy for control of dynamic balance of growth and development of the nervous system.3 During the normal development of the vertebrate nervous system, up to 50% or more of many types of neurons die soon after they form synaptic connections with their target cells.3 This massive cell death is thought to reflect the failure of these neurons to obtain adequate amounts of specific neurotrophic factors that are produced by the target cells and that are required for the neurons to survive.4 The cell actively participates in this "suicidal" process and destroys itself.5
Apoptotic cell death is initiated under certain conditions in the adult nervous system. After adrenalectomy and steroid replacement, a sudden withdrawal of glucocorticoids results in widespread apoptosis of the hippocampal dentate granule cells.6 7 Furthermore, insults such as ischemia or excitotoxicity may induce apoptosis in the mature nervous system.8 9 10 11 Apoptosis can be activated by relatively mild stimuli. For example, while exposure of murine P-815 cells to temperatures between 43°C and 44°C induces apoptosis, exposure to higher temperatures induces necrosis.12 Cell death changes from apoptosis to necrosis above a critical heat load.12
Cellular necrosis is induced by global/forebrain ischemia13 14 15 and focal ischemia16 17 in rats; after middle cerebral artery (MCA) occlusion, selective neuronal necrosis evolves into infarction.18 19 Although cellular necrosis has been characterized as a function of duration of cerebral ischemia,18 20 to our knowledge there are no data about the effectors of apoptosis in response to mild or severe ischemia-reperfusion stimuli and the processes that determine this mode of cell death. DNA fragmentation is associated with apoptosis and can be identified with the use of a molecular biological immunohistochemical method.21 Since extensive DNA fragmentation is an important characteristic of apoptosis, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells.
The present study details the number and anatomic distribution of apoptotic cells exhibiting DNA fragmentation in the rat brain after 10 to 120 minutes of MCA occlusion. These data show that cellular DNA fragmentation is induced by mild to severe ischemic damage (10 to 120 minutes) and increases as a function of time of MCA occlusion. In addition, the topographical distribution of apoptotic cells exhibiting DNA fragmentation accompanies neuronal necrosis, and apoptotic cells exhibiting fragmentation are primarily localized to the inner boundary zones of the infarct.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Experiments were performed on 32 male Wistar rats weighing 260 to 300 g. We induced focal cerebral ischemia using a method of intraluminal vascular occlusion.17 Briefly, rats (n=24) were anesthetized with 3.5% halothane and maintained with 1.0% halothane in 70% N2O and 30% O2 with the use of a face mask. The rectal temperature was controlled at 37°C with a feedback-regulated water heating pad. Approximately 18.0 to 19.0 mm of 4-0 surgical nylon suture (with its tip rounded by heating near a flame) was inserted from the right external carotid artery and advanced into the internal carotid artery until it blocked the origin of the MCA for 10, 20, 30, 60, 90, and 120 minutes (n=4 per time point).
All rats with right MCA occlusion exhibited focal neurological deficits, characterized by failure to extend the left forepaw. Four rats served as a sham-operated control in which a 15-mm-long nylon monofilament was inserted into the internal carotid artery for 2 hours. This length of nylon monofilament was too short to occlude the MCA. These rats did not exhibit left-sided neurological deficits. Four normal rats served as a control for in situ detection of DNA fragmentation.
Experimental rats (n=32) were given an overdose of ketamine and xylazine and killed at 48 hours of reperfusion after withdrawing the intraluminal suture. Rat brains were fixed by transcardial perfusion with heparinized saline, followed by perfusion and immersion in 10% buffered formalin phosphate. A coronal section at the level of the anterior commissure22 was obtained from each rat with the use of a rodent brain matrix, and formalin-fixed, paraffin-embedded coronal slides (5 µm) were cut.
We used a molecular biological-histochemical system, a terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method (ApopTag kit, Oncor), for sensitive and specific staining of DNA fragmentation and apoptotic bodies,21 and we identified the number of apoptotic cells exhibiting DNA fragmentation in both the ipsilateral and contralateral hemispheres using a light microscope. Cells containing apoptotic bodies are referred to as apoptotic cells.21 23 24 The TUNEL method is based on the specific binding of TdT to 3'-OH ends of DNA and the ensuing synthesis of a polydeoxynucleotide polymer. Briefly, after deparaffinizing brain sections and digesting protein in section using proteinase K and then quenching endogenous peroxidase activity with 2% H2O2 in PBS, we placed slides in equilibration buffer and then in working-strength TdT enzyme, followed by working strength stop/wash buffer. After two drops of anti-digoxigenin-peroxidase was applied to the slides, peroxidase was detected with diaminobenzidine. Negative controls were performed with distilled water for TdT enzyme in the preparation of working-strength TdT. The labeling target of the TUNEL method was the new 3'-OH DNA ends generated by DNA fragmentation, which was typically localized in morphologically identifiable nuclei and apoptotic bodies. The direct immunoperoxidase detection of digoxigenin-labeled DNA in thin sections of fixed tissue allowed analysis of scattered apoptotic cells at anatomic locations after MCA occlusion. In contrast, normal nuclei, which had relatively insignificant numbers of DNA 3'-OH ends, do not stain with the kit.23 Coronal sections stained with the TUNEL method were also counterstained with hematoxylin as a nuclear stain. Adjacent sections of tissue were stained with hematoxylin and eosin (H&E) to identify the necrotic cells and to correlate the anatomic distribution of necrotic and apoptotic cell death in rat brain subjected to MCA occlusion. Histological features used to identify the ischemic lesion included vacuolation (sponginess) of the neuropil, diffuse pallor of the eosinophilic background, and alterations in the shape and stainability of both neuronal perikarya and astrocytic nuclei. Necrotic neurons were identified as having pyknotic nuclei and an eosinophilic cytoplasm (red neuron) or lacking cellular structures (ghost neuron).25
Double staining was performed on sections from three rats subjected to 2 hours of MCA occlusion and 48 hours of reperfusion with the use of the TUNEL method and the avidin-biotin complex method26 and a peroxidase substrate kit for cellular identification. The sections were stained for identification of neurons and astrocytes with neuron-specific enolase (Dako) and glial fibrillary acidic protein (Dako), respectively. Briefly, after the nonspecific reaction was blocked with 5% horse serum, sections were washed in PBS and incubated with specific antibodies. The sections were again washed in PBS and incubated in biotinylated horse anti-mouse IgG (absorbed in rat serum; Vector) and in avidin-biotin complex (ABC kit, Vector). Peroxidase was demonstrated with a VIP kit (Vector).
Paired t tests were performed to detect differences of apoptotic cells between the rats subjected to MCA occlusion and sham-operated rats. Data are presented as mean±SD.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The negative controls in which distilled water with working-strength TdT was used, the control animals (Fig 1a
), and the contralateral hemisphere from the animals
subjected to ischemia exhibited no background labeling.
However, when the TUNEL method of staining was used, DNA fragmentation
was found in 0 to 3 cells within 5-µm-thick coronal sections in each
hemisphere of all normal rats and sham-operated rats and in the
contralateral hemisphere of ischemic rats. Necrotic cells were
not detected in normal and sham-operated rats or in the contralateral
hemisphere of ischemic rats.
|
Fig 2 illustrates the induction of apoptotic cells
exhibiting DNA fragmentation after 10 to 120 minutes of MCA occlusion
and 48 hours of reperfusion. The top panel of Fig 2 depicts an overview
of the topographic distribution of apoptotic cells and necrotic
neurons. The bottom panel of Fig 2 shows the numbers of apoptotic cells
(P<.01 at all time points 
20 minutes) compared with sham
animals. A progressive increase of area of distribution and numbers of
apoptotic cells was found with increasing durations of ischemia
and was accompanied by an increasing area of neuronal necrosis.
|
After 10 to 20 minutes of ischemia, selective and focal
neuronal necrosis was confined to the preoptic area and the striatum
(Fig 2
, top panel). Scattered apoptotic cells were present among
normal cells or red and ghost neurons in the same regions of the
neuronal necrosis. The number of apoptotic cells (10 to 20 per section)
increased significantly (P<.01) after 20 minutes of MCA
occlusion compared with control rats. With H&E staining and light
microscopic analysis (x300 field), apoptosis was
recognized by the presence of rounded or oval bodies (apoptotic
bodies), typically intensely dark purple-blue masses (2 to 10 per cell)
and variable in size (Fig 1c and 1e). The TUNEL method was far more
sensitive in detecting apoptotic bodies than H&E staining. With the use
of the TUNEL method, apoptotic bodies were clearly evident and were
stained dark brown for DNA breaks. Most apoptotic cells contained
multiple brown apoptotic bodies (2 to 25 per cell; Fig 1b, 1d, and 1f).
Apoptotic bodies were round or oval and varied considerably in
size.
After 30 to 60 minutes of ischemia, morphological evidence of
infarction was observed in the preoptic area and the striatum; in
contrast, selective and focal neuronal necrosis was observed in the
cortex (Fig 2, top panel). Compared with 10 to 20 minutes of
ischemia, scattered apoptotic cells (30 to 60 per section)
expanded into the cortex and were detected with both H&E staining (Fig 1c) and TUNEL method staining (Fig 1d).
The number of apoptotic cells markedly increased (70 to 200 per
section) in the infarct after 90 to 120 minutes of ischemia
(Fig 2, top panel). Scattered apoptotic cells were present
throughout the lesion area; however, groups of contiguous cells
exhibited DNA fragmentation and were primarily localized to the inner
boundary zone of the ischemic infarction (Fig 1e and 1f).
Mitotic cells at anaphase or telophase and neutrophils exhibit multiple
masses and are clearly distinguished from apoptotic cells with the use
of the in situ end-labeling of fragmented DNA method.23 24
Fig 1g and 1h show cells at anaphase present after 90 minutes of
MCA occlusion. Fig 1i and 1j show neutrophils present in the area
of cellular necrosis. Even with H&E staining, cells at anaphase and
neutrophils can be distinguished from apoptotic cells. In cells at
anaphase, the chromosomes move from the equatorial plate toward the
poles of the cell and always display rodlike shapes (Fig 1g) instead of
the round or oval shapes of apoptotic bodies (Fig 1b through 1f).
Neutrophils display three to five distinct lobes joined by thin strands
of chromatin, with the cytoplasm staining eosinophilic pink (Fig 1i).
However, with the TUNEL method and hematoxylin counterstaining,
apoptotic bodies stain brown (Fig 1b, 1d, 1f, and 1j), but cells at
anaphase (Fig 1h) and neutrophils (Fig 1j) stain blue. The lobes of
neutrophils are located within the nuclear membrane and do not protrude
to the cell surface (Fig 1i and 1j).
Double staining with the TUNEL method with different specific
antibodies revealed that most apoptotic cells were neurons (
90% to
95%, Fig 1k and 1l) and some were astrocytes (5% to 10%, Fig 1m and 1n). A few apoptotic endothelial cells (<1%) were
detected with H&E staining (Fig 1o) and with the TUNEL method and
hematoxylin counterstaining (Fig 1p).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To our knowledge, these are the first data detailing the anatomic distribution of apoptotic cells in response to MCA occlusion ranging from 10 minutes to 2 hours with the use of morphological criteria of apoptotic bodies identified by TUNEL staining. Apoptotic cells appeared concomitantly and mirrored the distribution of neuronal necrosis after MCA occlusion.18 19 20 Scattered apoptotic cells were present in the preoptic area and the striatum after short durations of ischemia (10 to 20 minutes) and then expanded to the cortex (30 to 60 minutes). However, groups of apoptotic cells were primarily present in the boundary zone of the ischemic core after a longer duration of ischemia (90 to 120 minutes).
There are three types of cell death distinct from necrosis that have been described in the nervous system from ultrastructural studies2 27 : apoptosis, autophagic cell death, and cytoplasmic cell death. Apoptotic cell death was originally found in the cervical visceromotor neurons.28 Autophagic cell death is characterized by the autophagic vesicles and is seen in the chick isthmo-optic nucleus after intraocular injection of colchicine.29 Cytoplasmic cell death occurs in embryonic chick ciliary ganglion cells30 and is characterized by dilation of the rough endoplasmic reticulum, swelling of mitochondria, and increased granularity of the nucleus at advanced stages. Morphological and ultrastructural changes are open to different interpretations, and the need for a biochemical marker to confirm a particular form of cell death is vital to delineation of the process of apoptosis.31 The detection of internucleosomal DNA fragmentation in situ with the typical apoptotic body morphology is a marker of apoptotic cell death.21 23 24 TUNEL staining is based on the detection of DNA strand breaks, which are abundantly present in apoptotic cells. In apoptotic bodies the chromatin is dense, fragmented, and packed into compact membrane-bound bodies together with randomly distributed cell organelles. The plasma membrane loses its characteristic architecture and shows extensive blebbing. It buds off projections so that the whole cell may split into several membrane-bound apoptotic bodies. We have recently obtained TUNEL staining data at various time points (from 0.5 hour to 28 days) after 2 hours of MCA occlusion.32 The numbers of apoptotic cells increased as early as 0.5 hour, peaked at 24 to 48 hours, and persisted for 4 weeks. The prolonged presence of apoptosis after the onset of ischemia suggests that apoptotic ischemic brain damage is a dynamic ongoing process. We have also obtained confirming evidence for apoptosis after MCA occlusion from electron microscopy33 and gel electrophoresis.32
The TUNEL method is based on the specific binding of TdT to 3'-OH ends
of DNA, which are abundantly present in apoptotic
cells.21 23 24 In necrotic cells lysosomal enzymes
generate mainly 5'-OH end groups of DNA.34 Although the
TUNEL method is based on the visualization of free 3'-OH end groups
present in DNA, the technique may detect 5'-OH ends of DNA. Wijsman
et al23 detailed that cellular debris and nuclear
"ghosts" showed faint, diffuse staining and indicated that since
in necrotic cells the DNA is degraded by release of lysosomal DNAse,
these cells incorporate the label as well. In necrotic cells we also
observed diffuse staining of cells. However, the TUNEL stain technique
to specifically identify apoptotic cells combines the biochemical
characteristic of DNA fragmentation with the characteristic apoptotic
morphology. The histological appearance of nuclei of
necrotic and apoptotic cells has been described.35 The two
modes of cell death, necrosis and apoptosis, each exhibit a
distinct morphology. The chromatin in nuclei of necrotic cells often
appears fairly uniformly compacted, and rupture of the nuclear membrane
and marginated chromatin masses are evident as small discrete masses
(karyorrhexis). Necrotic cells do not exhibit apoptotic cells. In
contrast, cells undergoing apoptosis exhibit apoptotic bodies
(Fig 1j). We emphasize that cells which exhibit apoptotic bodies with
TUNEL staining are apoptotic cells and not necrotic cells. However, we
cannot exclude the possibility that DNA fragmentation can occur without
the morphological changes associated with apoptosis, ie,
apoptotic bodies.
Both neutrophils and mitotic cells at anaphase or telophase may be
confused with apoptotic cells when analyzed with H&E under low
magnification of the light microscope. However, with the TUNEL method
and hematoxylin counterstaining and with the use of morphological
criteria, apoptotic cells are distinctly identified (Fig 1h and 1j).
The induction of apoptosis versus necrosis is determined by the severity of the insult after hyperthermic stimuli.12 36 In the case of in vitro hyperthermic cell damage, heating murine P-815 cells at 43°C or 44°C for 30 minutes causes rapid and exclusive enhancement of apoptosis, whereas heating the same cells at 46°C or 47°C causes massive necrosis.12 After 45°C heating, both apoptosis and necrosis are increased. Duvall and Wyllie37 have suggested that mild membrane damage caused by injurious agents might allow an influx of calcium sufficient to trigger apoptosis but is insufficient to extensively activate phospholipases with resultant necrosis. Although apoptosis can be activated by a relatively mild heating stimulus, we demonstrate that apoptosis is induced by mild and severe ischemia-reperfusion stimuli. Similar studies have shown that apoptosis occurs during the reperfusion phase after various durations of renal ischemia.38 In rat renal tissues, increasing periods of arterial clamping increase numbers of apoptotic cells from 3 to 5 per section (5-minute ischemia) to 30 to 35 per section (45-minute ischemia). The role played by the intensity of ischemia in determining the induction of apoptosis is not understood.
The perifocal or "penumbral" zone has a more subtle deterioration of cellular metabolism after focal cerebral ischemia.18 19 Essentially, this zone comprises all tissues that have a cerebral blood flow that is sufficiently reduced to put the cells "at risk." Thus, if recirculation is not achieved and pharmacological protection is not instituted, the cells in the penumbral zone will die and the penumbral zone will become part of the final infarction.39 The anatomic distribution of apoptotic cells as a function of duration of ischemia suggests that at the penumbral border, cells are not so rapidly and severely damaged that they can undergo an appropriate cell death rather than undergo necrosis. In a solid organ, cells that die by apoptosis are not going to damage their neighbors. It is also possible that neurons begin to die by apoptosis and then succumb to necrosis as the surrounding environment becomes more toxic. The distribution of apoptotic cells accompanies neuronal necrosis in brain after focal cerebral ischemia ranging from 10 minutes to 2 hours. This suggests that apoptosis is initiated by all ischemia-reperfusion stimuli that cause neuronal necrosis. Durations of ischemia of less than 90 minutes in this model of focal cerebral ischemia have been associated with a reperfusion window for salvage of penumbral tissue from necrosis,19 and these durations of ischemia may also be associated with the reperfusion window of apoptosis. Thus, reperfusion after a duration of ischemia less than 90 minutes results in some protection of the tissue, with a smaller necrotic lesion and fewer numbers of apoptotic cells. However, ischemia for 90 minutes or greater generates an infarct nearly identical to that found after permanent MCA occlusion.40 At 90 minutes of ischemia and above, clusters of apoptotic cells are primarily localized to the inner border zone of the infarct compared with the scattered distribution of apoptotic cells among the scattered areas of selective neuronal necrosis and focal infarct found at shorter durations of ischemia (10 to 60 minutes). It is interesting to note that the localization of the apoptotic cells at the inner necrotic boundary after a prolonged duration of ischemia gives the appearance of a process that expands the boundaries and the dimension of the lesion. This also suggests that apoptosis may contribute to the final infarct volume.
More than a decade ago, Siesjö41 suggested that free radicals generated during incomplete ischemia or during recirculation after complete ischemia significantly contribute to the final brain necrotic damage incurred. The evidence for a free radical component of the ischemic necrosis is that ischemic cell damage is reduced in animals treated with drugs whose effect is to scavenge free radicals42 43 or in transgenic mice in which the human copper-zinc superoxide dismutase is overexpressed.44 45 46 The overexpression of copper-zinc superoxide dismutase inhibits apoptosis.47 Hockenbery et al48 proposed that the bcl-2 proto-oncogene inhibits most types of apoptotic cell death, implying a regulation of an antioxidant pathway at sites of free radical generation. In neuronal cells, the expression of bcl-2 is associated with a marked inhibition of neuronal cell death not only when apoptosis is induced by serum and growth factor withdrawal but also when neuronal cell death is induced by several other means, including the addition of membrane-peroxidizing agents (eg, hydrogen peroxide or tert-butylhydroperoxide) and free radical–inducing agents (eg, menadione or 6-hydroxydopamine).47 Additionally, there is new evidence that bcl-2 expression also inhibits nonapoptotic cell death. Both buthionine sulfoximine and diethylmaleate produce necrotic cell death,49 yet the expression of bcl-2 clearly increases cell survival. Expression of bcl-2 in hypothalamic neuronal cells leads to a decrease in the generation of reactive oxygen species during apoptosis and necrosis.47 These studies suggest that both necrosis and apoptosis may be mediated directly by reactive oxygen species. The distribution of the apoptotic cells after ischemia-reperfusion is consistent with the hypothesis of free radical–mediated cell damage.
Implicit in our data that apoptosis is an active process is the possibility of its selective regulation. The capacity to control apoptosis in ischemic tissues may have beneficial consequences.
|
Acknowledgments |
|---|
This study was supported in part by grants PO1 NS23393 and RO1 NS29463 from the National Institute of Neurological Disorders and Stroke. The authors are grateful to Dr Gary J. Gibson for helpful comments, Qin Wang for technical assistance, and Denice Janus for secretarial support.
|
Footnotes |
|---|
Reprint requests to Michael Chopp, PhD, Department of Neurology, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI 48202.
Received September 20, 1994; revision received January 12, 1995; accepted March 30, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Kerr JFR, Wyllie AH, Currie AR. Apoptosis, a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239-245. [Medline] [Order article via Infotrieve]
2. Server AC, Mobley WC. Neuronal cell death and the role of apoptosis. In: Tomei LD, Cope FO, eds. Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1991:263-278.
3.
Raff MC, Barres BA, Burne JF, Coles HS, Ishizaki Y,
Jacobson MD. Programmed cell death and the control of cell
survival: lessons from the nervous system. Science. 1993;262:695-700.
4.
Martin DP, Schmidt RE, de Stefano PS, Lowry OH, Carter
JG, Johnson EM. Inhibitors of protein synthesis and
RNA synthesis prevent neuronal death caused by nerve growth factor
deprivation. J Cell Biol. 1988;106:829-844.
5. Sen S. Programmed cell death: concept, mechanism and control. Biol Rev. 1992;67:287-319. [Medline] [Order article via Infotrieve]
6. Sloviter RS, Dean E, Neubort S. Electron microscopic analysis of adrenalectomy-induced hippocampal granule cell degeneration in the rat: apoptosis in the adult central nervous system. J Comp Neurol. 1993;330:337-351. [Medline] [Order article via Infotrieve]
7. Sloviter RS, Sollas AL, Dean E, Neubort S. Adrenalectomy-induced granule cell degeneration in the rat hippocampal dentate gyrus: characterization in vivo model of controlled neuronal death. J Comp Neurol. 1993;330:324-336. [Medline] [Order article via Infotrieve]
8. Kure S, Tominaga T, Yoshimoto T, Tada K, Narisawa K. Glutamate triggers internucleosomal DNA cleavage in neuronal cells. Biochem Biophys Res Commun. 1991;179:39-45. [Medline] [Order article via Infotrieve]
9. Roberts-Lewis JM, Marcy VR, Zhao Y, Fedora T, Lewis ME, Siman R, Vaught JL. The apoptosis inhibitor: aurintricarboxylic acid prevents excitotoxin-induced neuronal death in vivo. Soc Neurosci Abstr. 1992;18:44. Abstract.
10.
Linnik MD, Zobrist RH, Hatfield MD. Evidence
supporting a role for programmed cell death in focal cerebral
ischemia in rats. Stroke. 1993;24:2002-2009.
11. Tominaga T, Kure S, Narisawa K, Yoshimoto T. Endonuclease activation following focal ischemic injury in the rat brain. Brain Res. 1993;608:21-26. [Medline] [Order article via Infotrieve]
12. Harmon BV, Corder AM, Collins RJ, Gobe GC, Allen J, Allan DJ, Kerr JFR. Cell death induced in a murine mastocytoma by 42-47 degrees C heating in vitro: evidence that the form of death changes from apoptosis to necrosis above a critical heat load. Int J Rad Biol. 1990;85:845-858.
13.
Pulsinelli WA, Brierley JB. A new model of
bilateral hemispheric ischemia in the unanesthetized
rat. Stroke. 1979;10:267-272.
14. Smith ML, Auer RN, Siesjö BK. The density and distribution of ischemic brain injury in the rat following 2-10 min of forebrain ischemia. Acta Neuropathol (Berl). 1984;64:319-332. [Medline] [Order article via Infotrieve]
15. Smith ML, Bendek G, Dahlgren N, Rosen L, Wieloch T, Siesjö BK. Models for studying long-term recovery following forebrain ischemia in the rat, II: a 2-vessel occlusion model. Acta Neurol Scand. 1984;68:385-401.
16. Tamura A, Graham DI, McCulloch J, Teasdale GM. Focal cerebral ischemia in the rat, II: regional cerebral blood flow determined by 14C-iodoantipyrine autoradiography following middle cerebral artery occlusion. J Cereb Blood Flow Metab. 1981;1:61-69. [Medline] [Order article via Infotrieve]
17.
Zea Longa E, Weinstein PR, Carlson S, Cummins R.
Reversible middle cerebral artery occlusion without craniectomy
in rats. Stroke. 1989;20:84-91.
18. Memezawa H, Minamisawa H, Smith ML, Siesjö BK. Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat. Exp Brain Res. 1992;89:67-78. [Medline] [Order article via Infotrieve]
19.
Memezawa H, Smith ML, Siesjö BK. Penumbral
tissues salvaged by reperfusion following middle cerebral artery
occlusion in rats. Stroke. 1992;23:552-559.
20.
Li Y, Chopp M, Garcia JH, Yoshida Y, Zhang ZG, Levine
SR. Distribution of the 72-kDa heat shock protein as a function
of transient focal cerebral ischemia in the rat.
Stroke. 1992;23:1292-1298.
21.
Gavrieli Y, Sherman Y, Ben-Sasson SA.
Identification of programmed cell death in situ via specific
labeling of nuclear DNA fragmentation. J Cell
Biol. 1992;119:493-501.
22. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 2nd ed. New York, NY: Academic Press, Inc; 1986.
23. Wijsman JH, Jonker RR, Keijzer R, Ven de Velde CJH, Cornelisse CJ, Van Dierendonck JH. A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J Histochem Cytochem. 1993;41:7-12. [Abstract]
24. Wood KA, Dipasquale B, Youle RJ. In situ labeling of granule cells for apoptosis-associated DNA fragmentation reveals different mechanisms of cell loss in developing cerebellum. Neuron. 1993;11:621-632. [Medline] [Order article via Infotrieve]
25. Graham DI. Hypoxia and vascular disorders. In: Adams JH, Duchen LW, eds. Greenfield's Neuropathology. 5th ed. New York, NY: Oxford University Press, Inc; 1992:153-268.
26. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981;29:577-580. [Abstract]
27. Clarke PGH. Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol. 1990;181:195-213. [Medline] [Order article via Infotrieve]
28.
O'Connor TM, Wyttenbach CR. Cell death in the
embryonic chick spinal cord. J Cell Biol. 1974;60:448-459.
29. Hornung JP, Koppel H, Clarke PGH. Endocytosis and autophagy in dying neurons: an ultrastructural study in chick embryos. J Comp Neurol. 1989;283:425-429. [Medline] [Order article via Infotrieve]
30.
Pilar G, Landmesser L. Ultrastructural
differences during embryonic cell death in normal and
peripherally derived ciliary ganglia. J
Cell Biol. 1976;68:339-356.
31. Zakeri ZF, Quaglino D, Latham T, Lockshin RA. Delayed internucleosomal DNA fragmentation in programmed cell death. FASEB J. 1993;7:470-478. [Abstract]
32. Li Y, Chopp M, Jiang N, Yao F, Zaloga C. Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab. 1995;15:389-397. [Medline] [Order article via Infotrieve]
33. Li Y, Sharov VG, Jiang N, Zaloga C, Sabbah TN, Chopp M. Ultrastructural and light microscopic evidence of apoptosis after middle cerebral artery occlusion in rat. Am J Pathol. In press.
34. Novikoff AB. Lysosomes: a personal account. In: Hers HG, Van Hoof F, eds. Lysosomes and Storage Diseases. New York, NY: Academic Press, Inc; 1973:2-41.
35. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251-306. [Medline] [Order article via Infotrieve]
36. Kerr JFR, Harmon BV. Definition and incidence of apoptosis: an historical perspective. In: Tomei LD, Cope FO, eds. Current Communications in Cell and Molecular Biology: Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1991;3:5-29.
37. Duvall E, Wyllie AH. Death and the cell. Immunol Today. 1986;7:115-119.
38. Schumer M, Colombel MC, Sawczuk IS, Gobe G, Connor J, O'Toole KM, Olsson CA, Wise GJ, Buttyan R. Morphologic, biochemical and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol. 1992;831-838.
39. Siesjö BK. A new perspective on ischemic brain damage? Prog Brain Res. 1993;96:1-9. [Medline] [Order article via Infotrieve]
40. Zhang RL, Chopp M, Chen H, Garcia JH. Temporal profile of ischemic tissue damage, neutrophil response, and vascular plugging following permanent and transient (2H) middle cerebral artery occlusion in the rat. J Neurol Sci. 1994;125:3-10. [Medline] [Order article via Infotrieve]
41. Siesjö BK. Cell damage in the brain: a speculative synthesis. J Cereb Blood Flow Metab. 1981;1:155-185. [Medline] [Order article via Infotrieve]
42.
Liu TH, Beckman JS, Freeman BA, Hogan EL, Hsu CY.
Polyethylene glycol-conjugated superoxide dismutase and catalase
reduce ischemic brain injury. Am J Physiol. 1989;256:H589-H593.
43.
Martz D, Rayos G, Schielke GP, Betz AL.
Allopurinol and dimethylthiourea reduce brain
infarction following middle cerebral artery occlusion in the
rat. Stroke. 1989;20:488-494.
44.
Kinouchi H, Epstein CJ, Mizui T, Carlson E, Chen SF,
Chan PH. Attenuation of focal cerebral ischemic injury
in transgenic mice overexpressing CuZn superoxide dismutase.
Proc Natl Acad Sci U S A. 1991;88:11158-11162.
45. Chan PH, Kinouchi H, Epstein CJ, Carlson E, Chen SF, Imaizumi S, Yang GY. Role of superoxide dismutase in ischemic brain injury: reduction of edema and infarction in transgenic mice following focal cerebral ischemia. Prog Brain Res. 1993;96:97-104. [Medline] [Order article via Infotrieve]
46. Yang G, Chan PH, Chen J, Carlson E, Chen SF, Weinstein P, Epstein CJ, Kamii H. Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia. Stroke. 1994;25:165-170. [Abstract]
47. Bredesen DE. Neuronal apoptosis: genetic and biochemical modulation. In: Tomei LD, Cope FO, eds. Apoptosis II: The Molecular Basis of Apoptosis in Disease. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1994;8:397-421.
48. Hockenbery DM, Oltvai Z, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxide pathway to prevent apoptosis. Cell. 1993;75:241-251. [Medline] [Order article via Infotrieve]
49.
Kane DJ, Saraflan TA, Anton R, Hahn H, Gralla E,
Valentine J, Ord T, Bredesen DE. Bcl-2 inhibition of neural
death: decreased generation of reactive oxygen species.
Science. 1993;262:1274-1277.
This article has been cited by other articles:
 |
|
 |
|
  B. R.S. Broughton, D. C. Reutens, and C. G. Sobey Apoptotic Mechanisms After Cerebral Ischemia Stroke, May 1, 2009; 40(5): e331 - e339. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Lampl, M. Boaz, R. Gilad, M. Lorberboym, R. Dabby, A. Rapoport, M. Anca-Hershkowitz, and M. Sadeh Minocycline treatment in acute stroke: An open-label, evaluator-blinded study Neurology, October 2, 2007; 69(14): 1404 - 1410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Inoue, D. P. Davis, J. C. Drummond, D. J. Cole, and P. M. Patel The combination of isoflurane and caspase 8 inhibition results in sustained neuroprotection in rats subject to focal cerebral ischemia. Anesth. Analg., May 1, 2006; 102(5): 1548 - 1555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kawaguchi, J. C. Drummond, D. J. Cole, P. J. Kelly, M. P. Spurlock, and P. M. Patel Effect of Isoflurane on Neuronal Apoptosis in Rats Subjected to Focal Cerebral Ischemia Anesth. Analg., March 1, 2004; 98(3): 798 - 805. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Aulak, T. Koeck, J. W. Crabb, and D. J. Stuehr Dynamics of protein nitration in cells and mitochondria Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H30 - H38. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-H. Yeh, Y.-C. Wang, Y.-C. Wu, J.-J. Chu, and P. J. Lin Continuous Tepid Blood Cardioplegia Can Preserve Coronary Endothelium and Ameliorate the Occurrence of Cardiomyocyte Apoptosis Chest, May 1, 2003; 123(5): 1647 - 1654. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Andreassi, N. Botto, A. Rizza, M. G. Colombo, C. Palmieri, S. Berti, S. Manfredi, S. Masetti, A. Clerico, and A. Biagini Deoxyribonucleic acid damage in human lymphocytes after percutaneous transluminal coronary angioplasty J. Am. Coll. Cardiol., September 4, 2002; 40(5): 862 - 868. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Garnier, J. Middelanis, A. Jensen, and R. Berger Neuroprotective Effects of Magnesium on Metabolic Disturbance in Fetal Hippocampal Slices After Oxygen-Glucose Deprivation: Mediation By Nitric Oxide System Reproductive Sciences, March 1, 2002; 9(2): 86 - 92. [Abstract] [PDF]
|
|
|
|
|
|
N. J. Solenski, C. G. diPierro, P. A. Trimmer, A.-L. Kwan, and G. A. Helms Ultrastructural Changes of Neuronal Mitochondria After Transient and Permanent Cerebral Ischemia Stroke, March 1, 2002; 33(3): 816 - 824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sairanen, O. Carpen, M.-L. Karjalainen-Lindsberg, A. Paetau, U. Turpeinen, M. Kaste, P. J. Lindsberg, and D. F. Cechetto Evolution of Cerebral Tumor Necrosis Factor-{alpha} Production During Human Ischemic Stroke Editorial Comment Stroke, August 1, 2001; 32(8): 1750 - 1758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-R. Schabitz, T. T. Hoffmann, S. Heiland, R. Kollmar, J. Bardutzky, C. Sommer, and S. Schwab Delayed Neuroprotective Effect of Insulin-Like Growth Factor-I After Experimental Transient Focal Cerebral Ischemia Monitored With MRI Stroke, May 1, 2001; 32(5): 1226 - 1233. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Nakajima, A. Ishida, M. S. Lange, K. L. Gabrielson, M. A. Wilson, L. J. Martin, M. E. Blue, and M. V. Johnston Apoptosis Has a Prolonged Role in the Neurodegeneration after Hypoxic Ischemia in the Newborn Rat J. Neurosci., November 1, 2000; 20(21): 7994 - 8004. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-R. Schabitz, C. Sommer, W. Zoder, M. Kiessling, M. Schwaninger, S. Schwab, and S. P. Finklestein Intravenous Brain-Derived Neurotrophic Factor Reduces Infarct Size and Counterregulates Bax and Bcl-2 Expression After Temporary Focal Cerebral Ischemia Editorial Comment Stroke, September 1, 2000; 31(9): 2212 - 2217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Stammberger, A. Gaspert, S. Hillinger, P. Vogt, B. Odermatt, W. Weder, and R. A. Schmid Apoptosis induced by ischemia and reperfusion in experimental lung transplantation Ann. Thorac. Surg., May 1, 2000; 69(5): 1532 - 1536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Li, M. D. Silva, C. H. Sotak, and M. Fisher Temporal evolution of ischemic injury evaluated with diffusion-, perfusion-, and T2-weighted MRI Neurology, February 8, 2000; 54(3): 689 - 689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. He, T. Yamawaki, S. Yang, A. L. Day, J. W. Simpkins, H. Naritomi, and W. I. Rosenblum Experimental Model of Small Deep Infarcts Involving the Hypothalamus in Rats : Changes in Body Temperature and Postural Reflex • Editorial Comment: Changes in Body Temperature and Postural Reflex Stroke, December 1, 1999; 30(12): 2743 - 2751. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Lipton Ischemic Cell Death in Brain Neurons Physiol Rev, October 1, 1999; 79(4): 1431 - 1568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Martin-Villalba, I. Herr, I. Jeremias, M. Hahne, R. Brandt, J. Vogel, J. Schenkel, T. Herdegen, and K.-M. Debatin CD95 Ligand (Fas-L/APO-1L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Mediate Ischemia-Induced Apoptosis in Neurons J. Neurosci., May 15, 1999; 19(10): 3809 - 3817. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Fujioka, T. Taoka, K.-I. Hiramatsu, S. Sakaguchi, and T. Sakaki Delayed Ischemic Hyperintensity on T1-Weighted MRI in the Caudoputamen and Cerebral Cortex of Humans After Spectacular Shrinking Deficit Stroke, May 1, 1999; 30(5): 1038 - 1042. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P. Yu, C. Yeh, U. Strasser, M. Tian, and D. W. Choi NMDA Receptor-Mediated K+ Efflux and Neuronal Apoptosis Science, April 9, 1999; 284(5412): 336 - 339. [Abstract] [Full Text]
|
|
|
|
 |
|
 Y. Soini, P. Paakko, and V-P. Lehto Histopathological Evaluation of Apoptosis in Cancer Am. J. Pathol., October 1, 1998; 153(4): 1041 - 1053. [Full Text] [PDF]
|
|
|
|
 |
|
 H Ikeda, Y Suzuki, M Suzuki, M Koike, J Tamura, J Tong, M Nomura, and G Itoh Apoptosis is a major mode of cell death caused by ischaemia and ischaemia/reperfusion injury to the rat intestinal epithelium Gut, April 1, 1998; 42(4): 530 - 537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Lieberthal, S. A. Menza, and J. S. Levine Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells Am J Physiol Renal Physiol, February 1, 1998; 274(2): F315 - F327. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Mackey, Y. Wu, R. Hu, J. A. DeMaro, M. F. Jacquin, G. K. Kanellopoulos, C. Y. Hsu, and N. T. Kouchoukos Cell Death Suggestive of Apoptosis After Spinal Cord Ischemia in Rabbits Stroke, October 1, 1997; 28(10): 2012 - 2017. [Abstract] [Full Text]
|
|
|
|
|
|
S. P. Butcher, D. C. Henshall, Y. Teramura, K. Iwasaki, and J. Sharkey Neuroprotective Actions of FK506 in Experimental Stroke: In Vivo Evidence against an Antiexcitotoxic Mechanism J. Neurosci., September 15, 1997; 17(18): 6939 - 6946. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Kerrigan, D. J. Zack, H. A. Quigley, S. D. Smith, and M. E. Pease TUNEL-Positive Ganglion Cells in Human Primary Open-angle Glaucoma Arch Ophthalmol, August 1, 1997; 115(8): 1031 - 1035. [Abstract] [PDF]
|
|
|
|
|
|
M. Tagaya, K.-F. Liu, B. Copeland, D. Seiffert, R. Engler, J. H. Garcia, and G. J. del Zoppo DNA Scission After Focal Brain Ischemia : Temporal Differences in Two Species Stroke, June 1, 1997; 28(6): 1245 - 1254. [Abstract] [Full Text]
|
|
|
|
|
|
S. Wagner, M. Tagaya, J. A. Koziol, V. Quaranta, and G. J. del Zoppo Rapid Disruption of an Astrocyte Interaction With the Extracellular Matrix Mediated by Integrin {alpha}6ß4 During Focal Cerebral Ischemia/Reperfusion Stroke, April 1, 1997; 28(4): 858 - 865. [Abstract] [Full Text]
|
|
|
|
|
|
R. Schmidt-Kastner, H. Fliss, A. M. Hakim, and M. D. Ginsberg Subtle Neuronal Death in Striatum After Short Forebrain Ischemia in Rats Detected by In Situ End-Labeling for DNA Damage Stroke, January 1, 1997; 28(1): 163 - 170. [Abstract] [Full Text]
|
|
|
|
|
|
J. B. Schulz, M. Weller, and T. Klockgether Potassium Deprivation-Induced Apoptosis of Cerebellar Granule Neurons: A Sequential Requirement for New mRNA and Protein Synthesis, ICE-Like Protease Activity, and Reactive Oxygen Species J. Neurosci., August 1, 1996; 16(15): 4696 - 4706. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-P. Haring, E. L. Berg, N. Tsurushita, M. Tagaya, G. J. del Zoppo, and M. J Eppihimer E-Selectin Appears in Nonischemic Tissue During Experimental Focal Cerebral Ischemia Stroke, August 1, 1996; 27(8): 1386 - 1392. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||