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

Abstract

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.

Keywords

Global ischemia Apoptosis Necrosis Delayed neuronal death Rat

The neuronal response to brain ischemia depends on the severity and duration of the ischemic insult as well as on intrinsic neuronal factors that impart selective vulnerability to specific brain regions (Scholz, 1953). Brief periods of global ischemia kill vulnerable neurons in the CA1 region of the hippocampus whereas longer intervals of vascular occlusion kill neurons in the cerebral cortex and corpus striatum as well (Ito et al., 1975; Pulsinelli et al., 1982a). Although seemingly undamaged, neurons in other brain regions such as the CA3 region of the hippocampus or the paramedian cerebral cortex undergo transient alterations as evidenced by reversible changes in their subcellular organelles (Petito and Pulsinelli, 1984aa, b ). The postischemic interval required for the apparent development of selective neuronal necrosis is inversely proportional to the severity or duration of the ischemic insult—the so-called maturation phenomena described by Ito et al. (Ito et al, 1975). This interval is especially prolonged in the CA1 neurons of the hippocampus (Ito et al., 1975; Pulsinelli et al, 1982a). The term delayed neuronal death has been applied to this process (Kirino, 1982).

During the past few years, the concept of cell death via apoptosis has become increasingly important. This mechanism of cell death is mediated by regulated pathways involving defined gene products rather than by direct injury to plasma membranes as occurs in necrosis (for reviews, see Wyllie et al., 1980; Kerr and Harmon, 1991; Majno and Joris, 1995). Apoptosis is the mode of cell death during embryogenesis or after physiological atrophy (Raff et al., 1993) but occurs in disease states as well. A number of studies suggest that focal or global cerebral ischemia also may cause neuronal apoptosis. The appearance of DNA ladders on gel electrophoresis of extracted nuclear DNA is characteristic of the oligonucleosomes in apoptotic cells. DNA ladders have been identified after ischemia of 12 or 20 minutes in the rat or 5 or 20 minutes in the gerbil (Heron et al., 1993; Ferrer et al., 1994; Sei et al., 1994; Charriaut-Marlangue et al., 1995;MacManus et al., 1995; Nitatori et al., 1995; Volpe et al., 1995). In addition, techniques that identify apoptotic oligonucleosomes in tissue sections label nuclei of postischemic neurons in the CA1 region (Ferrer et al., 1994; Sei et al., 1994; MacManus et al., 1995; Nitatori et al., 1995; Volpe et al., 1995). Similar findings have been reported in focal ischemia as well (Linnik et al., 1993; MacManus et al., 1994; Li et al., 1995).

A discrepancy exists between the above biochemical studies and virtually all morphologic studies that examine the sequential changes of cell death in global ischemia. As outlined by Deshpande et al. (1992), none show evidence of neuronal apoptosis. In an attempt to resolve the apparent discrepancy between morphological studies showing necrosis and biochemical studies demonstrating, or suggesting, apoptosis, we subjected rats to 10 minutes of transient forebrain ischemia followed by 1 to 14 days of reperfusion. Brains were examined for the presence of apoptosis by light microscopy, in situ detection of DNA oligonucleosomes, and increased transcriptional and translational activity of p53, one of several genes involved in apoptosis (Yonish-Rovach et al., 1991). Preliminary reports have been published (Petito et al., 1996, Torres-Munoz et al., 1996).

MATERIALS AND METHODS

Adult male Wistar rats, weighing between 200 and 250 g, were subjected to four-vessel occlusion as previously described (Pulsinelli and Brierley, 1979). Under inhalation anesthesia, the vertebral arteries were cauterized, loose ligatures were placed around the common carotid arteries, and a suture was placed under the posterior neck muscles. After an overnight fast, the common carotid arteries were occluded and the neck ligature tightened in the awake hand-restrained animal. Animals were accepted for the experiment if unconsciousness, pupillary dilatation, and loss of righting reflexes developed and persisted during ischemia and if seizures were absent. Body temperature was maintained at 37±0.5°C during ischemia and until the animal regained thermal homeostasis after cerebral recirculation. After 10 minutes, the clasps and neck suture were removed and the animals killed 1, 2, 3, 5, and 14 days later by perfusionfixation with 4% freshly prepared paraformaldehyde in 0.1 mol/L phosphate buffer. Brains were removed 2 hours later or overnight and stored in 0.1 mol/L phosphate buffer for 2 to 4 days before processing.

A coronal slice from the level of the mid-dorsal hippocampus, together with a segment of small intestine, were dehydrated, embedded in paraffin, and stained with hematoxylin-eosin in 5-μm sections. The term ischemic cell change (ICC) was used to describe dead neurons with cytoplasmic eosinophilia and nuclear pyknosis. Their number was evaluated in one 400× microscopic field of the CA1 region in the mid-dorsal hippocampus and expressed as a percentage of all neurons in that field.

In situ end-labeling (ISEL) of DNA was performed using terminal deoxynucleotidyl-transferase (Gavrieli et al., 1992; Gold et al., 1993) obtained in kit form (ApopTag kit; Oncor Laboratories, Gaithersburg, MD, U.S.A.). Sections were deparaffinized, digested, and incubated at 37°C with dATP, dCTP, dGTP, and digoxigenin (DIG)-labeled dUTP. After thorough washing, sections were incubated with anti-DIG conjugate (ApopTag kit) and hydrogen peroxide (H₂O₂) and 3,3′-diaminobenzidine. Pretreatment of sections with DNAase, and the presence of labeled nuclei in the apical villae of the small intestine in the experimental sections, served as a positive control for the enzymatic procedures; omission of the enzyme served as negative controls. The number of ISEL-labeled cells was expressed as a percentage of all neurons and of all neurons with ICC in one high power field (400×) of CA1 neurons in the mid-dorsal hippocampus. All attempts were made to use the same region of mid-dorsal hippocampus as was used for the hematoxylin-eosin evaluation.

Combined ISEL and immunohistochemistry for cell-specific markers was performed as previously described (Petito and Roberts, 1995). Antibodies used included those directed against glial fibrillary acidic protein for astrocytes (1:1000 dilution, Dako Corporation, Carpenteria, CA, U.S.A.); isolectin B4 for microglia (40 μg/mL, Sigma, St. Louis, MO, U.S.A.); transferrin for oligodendrocytes (Benkovic and Connor, 1993) (1:1000 dilution, Cappel Labs., Durham, NC, U.S.A.), and SMI 31 and 33 for phosphorylated and nonphosphorylated neurofilaments (1:2000 dilution, Sternberger Monoclonals, Inc., Baltimore, MD, U.S.A.). Chromagens included diaminobenzidine (brown), VIP (purple), and alkaline phosphatase-red. With glial fibrillary acidic protein, transferrin and SMI, ISEL (diaminobenzidine, brown) preceded immunohistochemistry (VIP, purple or alkaline phosphatase-red); with isolectin B4, ISEL (VIP, purple) followed immunohistochemistry (diaminobenzidine, brown).

Immunoreactivity for p53 antigen was examined on deparaffinized sections using overnight incubation with anti-p53 monoclonal antibodies (1:50 dilution, Dako Corporation) after 10-minute exposure to microwave radiation in citrate buffer. This was followed by sequential incubation with biotinylated secondary antibody, the avidin-biotin complex, and H₂O₂-diaminobenzidine. A p53-expressing glioblastoma multiforme and ovarian carcinoma were used as a positive control. Omission of the primary antibody served as a negative control.

In situ hybridization and Northern blot analysis for p53 used a digoxigenin-labeled cRNA-probe, prepared by transcription of the commercially available wt-p53 cDNA (Oncogene Science, Uniondale, NY, U.S.A.) according to the Boehringer Mannheim Dig-RNA labeling kit (SP6/T7) protocol. Frozen and formalin-fixed paraffin embedded ovarian carcinoma and a p53-secreting cell line were used as positive controls. RNA stability was confirmed by hybridization using digoxigenin labeled oligo-poly-T as probe. Negative controls were performed by using sense riboprobes for p53; omission of probe was used as background control. Deparaffinized sections of rat brain and intestine were rehydrated and pretreated with 0.2 mol/L Hcl for 20′ at room temperature; digested with proteinase K (10 μg/mL in 50 mmol/L Tris 20 mmol/L MgCl₂ buffer) for 10′ at room temperature; and postfixed in 4% paraformaldehyde in phosphate buffered saline. The sections were washed, incubated in prehybridization solution at 42°C for 1 hour, drained, and incubated at 42°C for 16 hours in hybridization solution plus probe. The coverslips were removed and sections were washed in 2× SSC, 1× SSC, and 0.1× SCC, 10′ each, at room temperature. The sections then were treated with 0.3% Triton X-100 containing 2% sheep whole serum in 100 mmol/L Tris-Cl, pH 7.5, 150 mmol/L NaCl for 1 hour at room temperature, drained, and covered again with 0.1% Triton X-100 and 1% normal sheep serum in Tris buffer containing 1:500 dilution of alkaline phosphatase-red-anti DIG conjugate for 2 hours at room temperature. Sections were rinsed and covered with Tris-HCP buffer pH 9.5, 50 mmol/L MgCl for 5′ at room temperature followed by developing solution prepared according to supplier instructions (Boehringer DIG DNA labeling and detection kit). Slides were washed, counterstained with methyl green, and mounted with Brand ACCU mounting medium.

Northern blot analysis for p53 mRNA was performed on pooled RNA extracted from three dewaxed, rehydrated, and homogenized 20-μm paraffin-embedded sections (Quiagen RNeasy kit). GAPDH cDNA DIG labeled probe was used as a positive control. Extracted RNA was electrophoresed in 1% agarose gel containing 1 × 3-[N-morpholino] propane sulfonic acid buffer, pH 7.0; transferred by capillarity to a nylon membrane; and cross linked with 1600 μjoules/cm² (UV light) (Sambrode et al., 1989). The RNA on nylon membrane was hybridized with the p53 DIG-labeled cRNA probe, using hybridization solution consisting of 50% Formamide 6× SSPE 5× Denhart's solution, 10% dextran sulfate, 0.5% SDS and 100 μg/mL herring sheared sperm DNA at 42°C for 16 hours. The membrane was washed twice in 2×, and 0.5× SSC containing 0.1% SDS, incubated in northern blocking solution in maleate buffer followed by a 2-hour incubation with 1:5,000 anti-DIG-alkaline phosphatase-red conjugate in northern blocking solution. The membrane was washed in maleate buffer for 15′ at room temperature and equilibrated in 100 mmol/L Tris-Cl pH 9.5, 100 mmol/L NaCl, and 50 mmol/L MgCl₂ buffer. The membrane was immersed in freshly prepared solution of 45 μL of NTB plus 35 μL of X-phosphate solution in 10 mL of same equilibrating buffer. The membrane was left in the dark for 30 minutes. When the signal became visible, the membrane was washed with sterile water to prevent over-development.

Evaluation

All observations, including light microscopy, ISEL, and molecular biology were performed without knowledge of the experimental conditions. Three to four animals were used for each reperfusion period. Sham-operated rats (n = 4) served as controls. The animal surgery, including brain perfusion, was performed in the laboratory of Drs. Pulsinelli and Nowak and the morphological and molecular biology studies in the laboratory of Dr. Petito.

RESULTS

Nuclei of positive controls, intestinal epithelial cells, and all nuclei in sections pretreated with DNase were intensely labeled by the ISEL technique and were unstained when terminal deoxynucleotidyl-transferase was omitted. As described in more detail below, ISEL was confined to those neurons with ICC. It was absent in normal neurons, in neurons with cytoplasmic condensations and in approximately half of all neurons displaying cytoplasmic eosinophilia and nuclear pyknosis at day 3 and 5. Labeling intensity diminished over time, and was faint and confined to neuronal cytoplasm at day 14.

The light microscopic characteristics of the CA1 region of the hippocampus were similar to prior reports in this model (Pulsinelli et al. 1984). At one day after ischemia, mild nuclear swelling was apparent when compared with control animals (Fig. 1A and B). In situ end-labeling was absent. At 2 days after ischemia (Fig. 1C), many neurons displayed focal cytoplasmic condensations and increased basophilia of both cytoplasm and nucleus. However, nuclear changes characteristic of early apoptosis, including peripheral margination of chromatin crescents or chromatin clumps, were absent. In situ end-labeling was absent. Glial changes, which included mild hyperchromasia of perineuronal glial nuclei and slight enlargement of astrocyte nuclei, were first apparent at this time.

Fig. 1.

CA1 neurons in controls (A) and at postischemic days 1(B), 2(C) and 3(D). Nuclei are mildly swollen at day 1(B) and mild increases in nuclear and cytoplasmic staining intensity is present at day 2(C). Focal cytoplasmic condensations (arrows), and neurons with shrunken cell bodies and pyknotic nuclei (open arrow) are present at day 3. Hyperchromatic perineuronal glial nuclei are noted at day 2 and 3. Hematoxylin-eosin, original magnification ×400.

At 3 days after ischemia, the number of neurons with ICC averaged 66% (Fig. 1D). Two of these animals had moderate to severe neuronal necrosis that represented 76% and 98% of the total number of neurons. This was associated with microglial proliferation and phagocytosis that at times obscured the underlying ICC. In situ end-labeling was present in 50% and 100% of the neurons showing ICC in these two animals and remained absent in neurons without eosinophilic change. The third 3-day animal had milder damage with only 11% of neurons displaying ICC. In situ end-labeling was absent in both the neurons with ICC as well as in the neurons that appeared undamaged (Fig. 2A and B).

Fig. 2.

CA1 neurons, postischemic day 3. (A) Approximately 30% of neurons are shrunken with eosinophilic cytoplasm and pyknotic nuclei. Some of the remaining neurons have focal cytoplasmic condensations but their nuclei appear undamaged. Hematoxylin-eosin. (B) Some of the shrunken neurons with pyknotic nuclei show In situ end-labeling (ISEL)-positivity but others (arrows) are ISEL-negative. Original magnification A: ×400; B: ×1000.

At 5 days after ischemia, the number of ICC neurons averaged 28%. At this point, the neurons with ICC were more shrunken and often showed nuclear karyorrhexis, a process in which pyknotic nuclei in dead cells break up into multiple irregular fragments. Microglial proliferation and neuronal phagocytosis was prominent. At 14 days, the number of eosinophilic neurons had diminished, their neurons shrunken; nuclei had lost their hyperchromasia and cytoplasmic eosinophilia was decreased. Neuronal ISEL was present in most of the residual neurons with ICC (Fig. 2A and B).

These results are summarized in Table 1. The total number of CA1 neurons in one 400× field averaged 95 in controls, 104 at day 1, 107 at day 2, 167 at day 3, 88 at day 5, and 86 at day 14. The apparent increases at days 1 through 3 are related to the fact that neuronal shrinkage allowed more neurons to be included in the 400× field. The apparent decreases at days 5 and 14 reflect the fact that many of the dead neurons were obscured or removed by phagocytic cells. The average percentage of neurons with ICC was 66% at day 3, 37% at day 5, and 28% at day 14. Ischemic cell change and ISEL were absent at day 1 and 2. In situ end-labeling only labeled ICC neurons, although it was not always present in these; the ICC labeling incidence averaged 50% at days 3 and 5 and 100% at day 14.

Table 1.

Temporal relationship between the evolution of ischemic neuronal cell death and in situ end labeling of fragmented DNA in CA1 neurons

		Postischemic day
	Control	1	2	3	5	14
Total Number of Neurons	95	104	107	167	88	86
% of Neurons showing ICC	0	0	0	66%	37%	28%
% of ICC neurons with ISEL	0	0	0	50%	50%	100%

ICC, ischemic cell change characterized as neurons with pyknotic nuclei and eosinophilic cytoplasm; ISEL, in situ end labeling. There were 3 to 4 animals in each group. Above values are the group means.

As expected in this model (Pulsinelli et al. 1984), eosinophilic neuronal cell death developed in the distal CA4-6 region and represented 15% of these neurons at postischemic day 1 and 2, 19% at day 3, and 12% at day 5. Their cytoplasm was slightly labeled by ISEL at day 1 but no labeling was present at subsequent periods. Mild increases in astrocytic nuclei and numbers of activated microglia accompanied this necrosis between day 1 and 5. Neurons in the CA3 region were unchanged from controls.

Rare cells with one or more central hyperchromatic nuclear bodies were present in control and postischemic hippocampus and were ISEL-positive. Some colabeled with transferrin on postischemic days 1 through 3, suggesting an oligodendroglial origin as described in preliminary reports (Petito and Torres-Munoz, 1996; Petito et al., 1996).

Fig. 3.

CA1 neurons, post-ischemic day 5. A. Severe neuronal necrosis is seen with hematoxylin-eosin stain. Residential neurons show mild nuclear swelling or cytoplasmic condensations. B. Most neurons display intense in situ end labeling (ISEL). Original magnification ×100.

p53 protein and mRNA were detected in the positive controls but were absent in control and postischemic rat brain (data not shown). Similarly, northern blot analysis for p53 mRNA was positive in the p53 secreting Cos7 cell line and in the paraffin-embedded and fresh frozen ovarian carcinoma, but p53 mRNA was not detected in control or post-ischemic rat brain (Fig. 4).

Fig. 4.

(A) Ethidium bromide-stained gel electrophoresis of total RNA isolated from lane 1: deparaffinized ovarian carcinoma; lane 2: fresh frozen ovarian carcinoma; lane 3: Cos-7 Cells; lane 4: rat brain, 3 days after ischemia; lane 5: rat brain, 1 day after ischemia; lane 6: rat brain, 5 days after ischemia; lane 7: rat brain, 14 days after ischemia; lane 8: rat brain, 2 days after ischemia; lane 9: rat brain, sham operated control. Lane M shows size RNA ladder markers. (B) Northern blot analysis. Total RNA isolated from positive controls and rat brain as above and blotted with digoxigenin-labeled cDNA probe for p53. p53 message is present in deparaffinized and fresh frozen ovarian carcinoma and COS-7 cells but absent in rat brain samples.

Review of prior material

Plastic-embedded sections of CA1 neurons subjected to 30 minutes of forebrain ischemia (Petito and Pulsinelli, 1984b) showed mild cytoplasmic swelling at 24 hours followed by cytoplasmic and nuclear shrinkage between 36 and 48 hours. Peripheral accumulations of nuclear chromatin or the single or multiple round dense chromatin masses in neurons with intact cytoplasm were absent. Paraffin-embedded sections from rats exposed to 2 or 10 minutes of forebrain ischemia (Petito et al., 1990; Petito and Halaby, 1993) also revealed mild cytoplasmic dilation that preceded the appearance of eosinophilic neurons. Morphological evidence of neuronal apoptosis was absent.

DISCUSSION

Specific morphological criteria identify apoptosis in tissue sections (Kerr, 1965; Wyllie et al., 1980; Kerr and Harmon, 1991; Majno and Joris, 1995). Initially, focal accumulations of dense chromatin masses develop at the nuclear periphery which leads to progressive nuclear shrinkage with or without fragmentation into multiple round chromatin masses. The nuclear changes are initiated by DNA fragmentation into 180 bp oligonucleosomal fragments as a result of an activation of a Ca⁺⁺Mg⁺⁺ endonuclease (Wyllie et al., 1984; Arends et al., 1990). Once the nuclear alterations have progressed, the cell body shrinks and may form cytoplasmic buds, in which are incorporated the chromatin masses. The final appearance of the apoptotic cell can be twofold: a single body with a shrunken cytoplasm, and a solitary round, pyknotic nucleus, or multiple small apoptotic bodies formed from breakup of the cytoplasmic buds. During this process, the plasma membrane retains its structural and functional integrity (Daryznkiewicz et al, 1992). Apoptosis can take place in the absence of protein or mRNA synthesis, may be preceded by abnormalities in mitochondrial function, and may develop in anucleate cells in the absence of DNA fragmentation (Schwartzman and Cidlowski, 1993; Hockenbery, 1995; Kane, 1995; Kroemer et al., 1995;). Its control is based not only on nuclear susceptibility but also on specific cytoplasmic regulators (Polunovsky et al., 1996). Necrosis differs from apoptosis in the early stages because loss of plasma membrane integrity is one of the initial changes and leads to cell swelling which may or may not be transient. Although DNA breaks and nuclear pyknosis develop in necrosis, they appear after membrane damage and cytoplasmic changes and are not accompanied by peripheral chromatin condensations (Arends, et al. 1990; Gold et al. 1994).

DNA fragmentation can be detected in tissue sections by ISEL (Gavrieli et al., 1992; Wijsman et al., 1993). Either terminal deoxynucleotidyl-transferase or DNA polymerase I add labeled nucleotide tails to the 3′-OH ends of the cleaved DNA strands. Both methods bind nucleotides to the random nicks and breaks that occur in DNA of necrotic cells as well as to the oligonucleosomes of apoptotic cells (Gold et al., 1994). As a result, ISEL positivity alone does not distinguish between necrosis and apoptosis, a finding noted by a number of studies (Gold et al. 1993, Wijsman et al. 1993; Ansari et al, 1993; Mundle and Raza, 1995). If the cytoplasm already displays irreversible changes of cell death such as eosinophilic necrosis, ISEL-positivity cannot distinguish between these two processes. According to the current definition of apoptosis, ISEL identifies an otherwise normal-appearing cell as apoptosis only if the membrane integrity of the cell is unaltered. For example, both apoptosis and necrosis develop in experimental myocardial infarct produced by coronary artery occlusion (Kajstura et al. 1996). DNA strand breaks, indicated by nuclear ISEL, developed with both processes, but plasma membrane permeability was confined to, and used to identify, necrotic rather than apoptotic myocytes.

Sequential light and electron microscopic studies of CA1 neurons subjected to global ischemia clearly show that the early chromatin changes of apoptosis are absent and that the cytoplasmic changes precede nuclear changes (Kirino, 1982; Kirino et al., 1984; Petito and Pulsinelli, 1984b; Deshpande et al., 1992; Nitatori et al., 1994). In our prior study of hippocampal neurons exposed to 30 minutes of forebrain ischemia (Petito and Pulsinelli, 1984b), transient swelling of mitochondria, Golgi cisterns, and endoplasmic reticulum appeared at 3 minutes; disaggregation of polyribosomes and loss of Golgi cisterns at 15, 30, and 120 minutes; and cytoplasmic swelling between 24 and 36 hours. Nuclear changes of apoptosis were absent. Other studies examining the cerebral cortex or striatum in global ischemia also show that cytoplasmic changes precede nuclear changes and that the early nuclear changes of apoptotic cells are not present (Brown and Brierley, 1972; Dodson et al., 1977; Ekström et al., 1982; Petito and Pulsinelli, 1984a; Dietrich et al., 1991).

The results of the present study suggests that ISEL in postischemic neurons results from nucleotide binding to DNA of necrotic cells rather than to oligonucleosomes of apoptotic cells. The nuclear changes associated with early apoptosis, such as chromatin condensations or peripheral chromatin aggregation, were absent. Rather, karyorrhexis was observed in the already dead neurons at postischemic day 5. Karyorrhexis is a relatively late process of necrosis, and one that required 2 days to develop in the present study. It is distinguished from apoptotic nuclear fragmentation into round chromatin masses by the fact that it develops in an already dead cell and by the fact its fragments are small and irregular (Majno and Joris, 1995). Second, ISEL was never present in prenecrotic neurons at days 1−3. If the CA1 death had occurred by apoptosis, ISEL-positivity should have been observed in neurons with normal appearing cell bodies. Lastly, ISEL appeared to be a late event, following rather than preceding cytoplasmic eosinophilia and shrinkage. It was absent in many neurons displaying early cytoplasmic changes associated with necrosis. The labeling intensity in the ICC neurons decreased and by 15 days after ischemia, it was often weak and confined to neuronal cytoplasm, a localization representing a presumed leak of broken DNA strands from the nucleus into the cytoplasm of the dead cell (Russell, 1983; Rink, 1995).

If morphology and ISEL do not identify neuronal apoptosis after global ischemia, what then accounts for the oligonucleosomes that clearly have been detected by the presence of DNA ladders on gel electrophoresis after global ischemia in rodents. Several factors may explain this apparent discrepancy.

First, some of the oligonucleosomal ladders detected after ischemic insults could result from apoptosis of glial cells or of neurons outside the hippocampus. In a preliminary study, we showed that there are actually a large number of glial cells that undergo apoptosis after 10 minutes of global ischemia (Petito et al., 1996). They were most numerous in brain regions such as the cerebral cortex where neuronal cell death is absent or minimal. Glial and microglial apoptosis occur in other conditions of brain injury (Kane, 1995; Petito and Roberts, 1995; Lin et al., 1996) and their presence in ischemia is therefore not unexpected.

Second, DNA ladders follow rather than precede cell death in type II programmed cell death (Schwartz et al, 1993; Zakeri et al., 1993; Lockshin and Zakeri, 1996), and similarities to that in postischemic CA1 neurons suggests that this cell death mechanism could occur after transient ischemia as well. Type II programmed cell death, or autophagy, is a second type of cell death during embryogenesis that is in addition to the more familiar apoptosis or Type I programmed cell death. Cytoplasmic abnormalities initiate autophagy, with increases in lysosomes and loss of rough endoplasmic reticulum that precede eventual cell collapse. Chromatin condensation and nuclear pyknosis are late events and are accompanied by DNA fragmentation into oligonucleosomes that appear as DNA ladders on gel electrophoresis. Type II programmed cell death has been described in models of hepatic toxicity (Fukuda et al., 1993) and also takes place in neurons or oligodendrocytes deprived of growth factors or treated with corticosteroids (Schwartz et al., 1993). Similarities between postischemic neuronal death and Type II programmed cell death include cytoplasmic alterations that develop before nuclear changes; ISEL positivity that occurs with or follows the appearance of eosinophilic necrosis; and DNA fragmentation into oligonucleosomes after cellular evidence of necrosis. In addition, accumulation of lysosomes and proteases precede cell death in hippocampal neurons after global ischemia (Nitatori et al., 1995) whereas inhibition of lysosomal proteases protects against delayed neuronal death produced by global ischemia (Lee et al, 1991; Hoffmann et al, 1992; Robert-Lewis et al, 1993; Palm et al, 1995).

Lastly, the current definitions of apoptosis and necrosis may be too restrictive, excluding other mechanisms, such as transitional forms between apoptosis and necrosis (MacManus et al, 1994; Porter-Callilau et al, 1995). These authors hypothesize that the orderly sequence of nucleic and cytoplasmic events that occurs in apoptosis is interrupted after DNA fragmentation into the oligonucleosomes seen as DNA ladders, and evolution into necrotic cell changes rather than into apoptotic cellular changes develops.

The exploration of potential genes that are directly involved in ischemic neuronal death is in its infancy. There is evidence of transcriptional and translational activity of early-immediate genes including c-fos, c-jun, and heat shock protein (Kogure and Kato, 1993; Nowak, et al., 1993; Estus et al., 1994; Akin et al., 1996). Expression of c-fos and c-jun have been associated with apoptosis in other model systems (Barrett and Preston, 1994), although available evidence indicates such responses are more likely to be involved in neuroprotective mechanisms after ischemia (Nowak et al, 1992; Sommer et al, 1995; Abe and Nowack, 1996). Members of the interleukin-converting enzyme gene family and p53 are common apoptosis-driving genes (Yonish-Rovach et al, 1991; Miura et al., 1993; Steller 1994; Hale et al, 1994; Sakhi et al., 1994; Deshmukh et al, 1996) that have been associated with focal ischemia (Crumrine et al, 1994; Li et al, 1994). Their role in global ischemia is less clear as shown by the absence of p53 protein and mRNA in the present study and of interleukin-converting enzyme in an earlier study by Bhat et al (1996). However, their absence does not exclude apoptosis because their participation is not necessarily required for apoptosis to take place (Hale et al, 1995; Vasilakos et al, 1995; Sadoul et al, 1996). Bax, another apoptosis-driving gene, has been identified by immunoreactivity in necrotic neurons after global ischemia produced by transient cardiac arrest (Krajewski et al, 1995).

The present study does not rule out other conditions in which ischemia could produce neuronal apoptosis. For example, apoptosis may be responsible for the late delayed, ongoing neuronal cell death that occurs weeks after the ischemic interval. Such changes have been described in the cerebral cortex, substantia nigra, and basal ganglia after focal or global ischemia (Tamara et al., 1990; Hara et al., 1993; Saji et al., 1994; Dietrich et al., 1995). If this late delayed neuronal death is caused by apoptosis, it is likely to be caused by indirect factors such as loss of trophic factors that are known to induce neuronal apoptosis (Barde, 1989; Deckwerth and Johnson, 1993), rather than as the direct result of the ischemic insult itself.

The severity of ischemia could be another variable determining whether postischemic neurons die by apoptosis or necrosis. Precedence for this is found in a number of conditions, such as hyperthermia (Harmon et al., 1990) and ultraviolet irradiation (Fukuda et al., 1993), in which a given insult causes apoptosis if mild but necrosis if severe. Indeed, Kerr's original observation of apoptosis was in a model of hepatic ischemia which produced necrosis as well as apoptosis in liver cells (Kerr, 1965). In the penumbral zone of cerebral infarcts, regional cerebral blood flow declines to only 20% of control values (Nagasawa and Kogure, 1989), an insult that is relatively mild when compared with the CBF reduction to 3% of control values during global ischemia (Pulsinelli et al., 1982b). Thus, neuronal apoptosis might be expected at the periphery of infarcts, and has been described in some studies (Li et al., 1995).

Acknowledgment: The authors thank Ms. Sharon Harris for her secretarial assistance.

Footnotes

Abbreviations used

References

Abe

Nowak

Jr. (1996a) Gene expression and induced ischemic tolerance following brief insults. Acta Neurobiol Exp 56:3–8

Akin

Liu

Hsu

(1996) Immediate early gene expression in response to cerebral ischemia. Stroke 27:1682–1687

Ansari

Coates

Greenstein

Hall

(1993) In situ end-labeling detects DNA strand breaks in apoptosis and other physiological and pathological states. J Pathol 170:1–8

Arends

Morris

Wyllie

(1990) Apoptosis: the role of the endonuclease. Am J Pathol 136:593–608

Barde

(1989) Trophic factors and neuronal survival. Neuron 2:1525–1534

Barrett

Preston

(1994) Apoptosis and cellular senescence: forms of irreversible growth arrest. In: Apoptosis II: The Molecular Basis of Apoptosis in Disease. ( Tomei

Cope

, eds). Plainview, NY, Cold Spring Harbor Laboratory Press, pp 253–281

Benkovic

Connor

(1993) Ferritin, transferrin, and iron in selected regions of the adult and aged rat brain. J Comp Neurol 338:97–113

Bhat

DiRocco

Marcy

Flood

Zhu

Dobrzanski

Siman

Scott

Contreras

Miller

(1996) Increased expression of IL-1B converting enzyme in hippocampus after ischemia: localization in microglia. J Neurosci 16:4146–4154

Mannheim

Boehringer

(1996) Genius System User's Guide For Membrane Hybridization. Boehringer Mannheim GmbH, Biochemica. Germaine-27, 52–54

10.

Mannheim

Boehringer

(1996) Nonradioactive In Situ Hybridization Application Manual 2 ed. Boehringer Mannheim GmbH, Biochemica, Germany, pp 126–131

11.

Brown

Brierley

(1972) Anoxic-ischemic cell change in rat brain: light microscopic and fine-structural observations. J Neurol Sci 16:59–84

12.

Charriaut-Marlangue

Marrgaill

Plotkine

Ben-Ari

(1995) Rapid communication: Early endonuclease activation following reversible focal ischemia in the rat brain. J Metabolism 15:385–388

13.

Crumrine

Thomas

Morgan

(1994) Attenuation of p53 expression protects against focal ischemic damage in transgenic mice. J Cereb Blood Flow Metab 14:887–891

14.

Darzynkiewicz

Bruno

Del Bino

Gorczyca

Hotz

Lassota

Traganos

(1992) Features of apoptotic cells measured by flow cytometry. Cytometry 13:795–808

15.

Deckwerth

Johnson

Jr (1993) Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor. J Cell Biol 123:1207–1222

16.

Deshmukh

Vasilakos

Deckwerth

Lampe

Shivers

Johnson

Jr. (1996) Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases. J Cell Biol 135:1341–1354

17.

Deshpande

Bergstedt

Linden

Kalimo

Wieloch

(1992) Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death. Exp Brain Res 88:91–105

18.

Dietrich

Halley

Valdes

Busto

(1991) Interrelationships between increased vascular permeability and acute neuronal damage following temperature-controlled brain ischemia in rats. Acta Neuropathol 81:615–625

19.

Dietrich

Lin

Mordecai

Y-T G

Green

Ginsberg

Busto

(1995) Effect of delayed MK-801 (Dizocilpine) treatment with or without immediate post-ischemic hypothermia on chronic neuronal survival after global forebrain ischemia in rats. J Cereb Blood Flow Metab 15:960–968

20.

Dodson

Chu

LWF

Welch

KMA

Achar

(1977) Acute tissue response to cerebral ischemia in the gerbil. J Neurol Sci 33:161–170

21.

Estus

Zaks

Freeman

Gruda

Bravo

Johnson

Jr. (1994) Altered gene expression in neurons during programmed cell death: Identification of c-jun as necessary for neuronal apoptosis. J Cell Biol 127:1717–1727

22.

Ferrer

Tortoise

Macaya

Sierra

Moreno

Munell

Blanco

Squier

(1994) Evidence of nuclear DNA fragmentation following hypoxia-ischemia in the infant rat brain, and transient forebrain ischemia in the adult gerbil. Brain Pathol 4:115–122

23.

Fukuda

Kojiro

Chiu

J-F

(1993) Demonstration of extensive chromatin cleavage in transplanted morris hepatoma 7777 tissue: apoptosis or necrosis? Am J Pathol 142:935–946

24.

Gavrieli

Sherman

Ben-Sasson

(1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501

25.

Gold

Schmied

Giegerich

Breitschopf

Hartung

Toyka

Lassmann

(1994) Differentiation between cellular apoptosis and necrosis by combined use of in situ tailing and nick translation techniques. Lab Invest 71:219–225

26.

Gold

Schmied

Rothe

Zishler

Breitschopf

Wekerle

Lassmann

(1993) Detection of DNA fragmentation in apoptosis: Application of in situ nick translation to cell culture systems and tissue sections. J Histochem Cytochem 41:1023–1030

27.

Hale

Harada

Sukamoto

(1993) Chronological atrophy after transient middle cerebral artery occlusion in rats. Brain Res 618:251–260

28.

Hara

Harada

Sukamoto

(1993) Chronological atrophy after transient middle cerebral artery occlusion in rats. Brain Res 618:251–260

29.

Harmon

Corder

Collins

Gobe

Allen

Allan

Keff

(1990) Cell death induced in a murine mastocytoma by 42–47°C heating in vitro: evidence that the form of death changes from apoptosis to necrosis above a critical heat load. Int J Radiat Biol 53:845–858

30.

Heron

Pollard

Dessi

Moreau

Lasbennes

Ben-Ari

Charriaut-Marlangue

(1993) Regional variability in DNA fragmentation after global ischemia evidenced by combined histological and gel electrophoresis observations in the rat brain. J Neurochem 61:1973–1976

31.

Hockenbery

(1995) Defining Apoptosis. Am J Pathol 146:16–19

32.

Hoffman

Nitsch

Scotti

Reinhard

Monard

(1995) The prolonged presence of glia-derived nexin, an endogenous protease inhibitor, in the hippocampus after ischemia-induced delayed neuronal neath. Neurosci 49:397–408

33.

Ito

Spat

Walker

Jr Klatzo

(1975) Experimental cerebral ischemia in Mongolian gerbils: I. Light microscopic observations. Acta Neuropathol 32:209–233

34.

Kajstura

Cheng

Reiss

Clark

Sonnenblick

Krajewski

Reed

Olivette

Anversa

(1996) Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest 74:86–107

35.

Kane

(1995) Commentary: redefining cell death. Am J Pathol 146:1–2

36.

Kerr

JFR

(1965) A histochemical study of hypertrophy and ischemic injury of rat liver with special reference to changes in lysosomes. J Pathol Bacteriol 90:419–435

37.

Kerr

JFR

Harmon

(1991) Definition and incidence of apoptosis: an historical perspective. In: Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, pp 5–29

38.

Kirino

(1982) Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 239:57–69

39.

Kirino

Tamara

Sago

(1984) Delayed neuronal death in the rat hippocampus following transient forebrain ischemia. Acta Neuropathol 64:139–147

40.

Kogure

Kato

(1993) Altered gene expression in cerebral ischemia. Stroke 24:2121–2127

41.

Krajewski

Mai

Krajewska

Sikorska

Mossakowski

Reed

(1995) Up-regulation of bax protein levels in neurons following cerebral ischemia. J Neurosci 15:6364–6376

42.

Kroemer

Petit

Zamzam

Vassar

J-L

Magnate

(1995) The biochemistry of programmed cell death. FASEB J 9:1277–1287

43.

Lee

Frank

Vanderklish

Arai

Lynch

(1991) Inhibition of proteolysis protects hippocampal neurons from ischemia. Proc Natl Acad Sci USA 88:7233–7237

44.

Chopp

Jiang

Yas

Zaloga

(1995) Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metabol 15:389–397

45.

Chopp

Zaloga

Niewenhuis

Gautam

(1994) p53-immunoreactive protein and p53 mRNA expression after transient middle cerebral artery occlusion in rats. Stroke 25:849–855

46.

Lin

Brodin

Farooque

Fonak Holtz

Wang

Olsson

(1996) Apoptosis and expression of bcl-2 after compression trauma to rat spinal cord. J Neuropath Exp Neurol 55:280–289

47.

Linnik

Zobrist

Pike

Whittemore

(1993) Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats. Stroke 24:2002–2009

48.

Lockshin

Zakeri

(1996) The biology of cell death and its relationship to aging. In: Cellular Aging and Cell Death ( Holbrook

Martin

Lockshin

, eds), New York, NY, Wiley-Liss, Inc., pp 167–180

49.

MacManus

Hill

Preston

Rasquinha

Walker

Buchan

(1995) Differences in DNA fragmentation following transient cerebral or decapitation ischemia in rats. J Cereb Blood Flow Metabol 15:728–737

50.

MacManus

Hill

Huang

Rasquinha

Xue

Buchan

(1994) DNA damage consistent with apoptosis in transient focal ischemic neocortex. Neuroreport 5:493–496

51.

Majno

Joris

(1995) Apoptosis, oncosis and necrosis: an overview of cell death. Am J Pathol 146:3–15

52.

Miura

Zhu

Rotello

Hartweig

Yuan

(1993) Induction of apoptosis in fibroblasts by II-1-β-converting enzyme, a mammalian homology of the C. elegans cell death gene (CED-3). Cell 75:653–660

53.

Mundle

Raza

(1995) The two in situ techniques do not differentiate between apoptosis and necrosis but rather reveal distinct patterns of DNA fragmentation in apoptosis. Lab Invest 72:611–613

54.

Nagasawa

Kogure

(1989) Correlation between cerebral blood flow and histological changes in a new rat model of middle cerebral artery occlusion. Stroke 20:1037–1043

55.

Nitatori

Sato

Waguri

Karasawa

Araki

Shibanai

Kominami

Uchiyama

(1995) Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J Neurosci 15:1001–1011

56.

Nowak

Jr. Osborne

Suga

(1993) Stress protein and protooncogene expression as indicators of neuronal pathophysiology after ischemia. In: Neurobiology of Ischemic Brain Damage—Progress in Brain Research, Vol. 96 ( Kogure

Hossmann

K-A

Siesjö

, eds). Amsterdam, Elsevier Science Publishers, pp 195–208

57.

Palm

Knuckey

Primiano

Spangenberger

Johanson

(1995) Cystatin C, a protease inhibitor, in degenerating rat hippocampal neurons following transient forebrain ischemia. Brain Res 691:1–8

58.

Pender

McCombe

Yoong

Nguyen

(1992) Apoptosis of αβ T lymphocytes in the nervous system in experimental autoimmune encephalomyelitis: its possible implications for recovery and acquired tolerance. J Autoimmunity 5:401–410

59.

Petito

Halaby

(1993) Relationship between ischemia and ischemic neuronal necrosis to astrocyte expression of glial fibrillary acidic protein. Int J Review Neurol 11:239–247

60.

Petito

Morgello

Felix

Lesser

(1990) Two patterns of reactive astrocytosis in post-ischemic rat brain. J Cereb Blood Flow Metabol 10:850–859

61.

Petito

Olarte

Nowak

Pulsinelli

(1996) Selective glial vulnerability to post-ischemic apoptosis [Abstract]. J Neuropath Exp Neurol 55:665

62.

Petito

Pulsinelli

(1984a) Sequential developmental of reversible and irreversible neuronal damage following cerebral ischemia. J Neuropathol Exp Neurol 43:141–153

63.

Petito

Pulsinelli

(1984b) Delayed neuronal recovery and neuronal death in rat hippocampus following severe cerebral ischemia: possible relationship to abnormalities in neuronal processes. J Cereb Blood Flow Metabol 4:194–205

64.

Petito

Roberts

(1995) Evidence of apoptotic cell death in HIV encephalitis. Am J Pathol 146:1121–1130

65.

Petito

Torres-Munoz

(1997) Morphological comparisons between apoptosis and necrosis in postischemic neuronal death. In: Frontiers in Cerebrovascular Disease: Mechanisms, Diagnosis and Treatment ( Robertson

Nowak

Jr , eds), Armonk, NY, pp 167–178

66.

Polunovsky

Ingbar

Peterson

Bitterman

(1996) Cell fusion to study nuclear-cytoplasmic interactions in endothelial cell apoptosis. Am J Pathol 149:115–128

67.

Pulsinelli

Brierley

(1979) A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10:267–272

68.

Pulsinelli

Brierley

Plum

(1982a) Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol 11:491–499

69.

Pulsinelli

Levy

Duffy

(1982b) Regional cerebral blood flow and glucose metabolism following treatment of forebrain ischemia. Ann Neurol 11:500–509

70.

Raff

Barres

Burne

Coles

Ishizaki

Jacobson

(1993) Programmed cell death and the control of cell survival: lessons from the nervous system. Science 262:695–700

71.

Rink

Fung

K-M

Trojanowski

Lee

VM-Y

Neugebauer

McIntosh

(1995) Evidence of apoptotic cell death after experimental traumatic brain injury in the rat. Am J Pathol 147:1575–1583

72.

Robert-Lewis

March

Zhao

Vaught

Siman

Lewis

(1993) Aurintricarboxylic acid protects hippocampal neurons from NMDA-and ischemia-induced toxicity in vivo. J Neurochem 61:378–381

73.

Russell

(1983) Internal disintegration model of cytotoxic lymphocyte-induced target damage. Immunol Rev 72:97–118

74.

Sadoul

Quiquerez

Martinou

Fernandez

Martinou

J-C

(1996) p53 protein in sympathetic neurons: cytoplasmic localization and no apparent function in apoptosis. J Neurosci Res 43:594–601

75.

Saji

Cohen

Blau

Wessel

Volpe

(1994) Transient forebrain ischemia induces delayed injury in the substantia nigra reticulata: degeneration of GABA neurons, compensatory expression of GAD mRNA. Brain Res 643:234–244

76.

Sakhi

Bruce

Sun

Tocco

Baudry

Schreiber

(1994) p53 induction is associated with neuronal damage in the central nervous system. Proc Natl Acad Sci USA 91:7525–7529

77.

Sambrook

Fritsch

Maniatis

(1989) Molecular Cloning. A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press 13:17–19

78.

Scholz

(1953) Selective neuronal necrosis and its topistic patterns in hypoxemia and oligemia. J Neuropath Exp Neurol 12:249–261

79.

Schwartz

Smith

Jones

MEE

Osborne

(1993) Do all programmed cell deaths occur via apoptosis? Proc Natl Acad Sci USA 90:980–984

80.

Schwartzman

Cidlowski

(1993) Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr Rev 14:133–146

81.

Sei

VonLubitz

Basile

Barner

Lin

RCS

Skolnick

Fossom

(1994) Internucleosomal DNA fragmentation in gerbil hippocampus following forebrain ischemia. Neurosci Lett 171:179–182

82.

Sommer

Gass

Kiessling

(1995) Selective c-jun expression in CA1 neurons of the gerbil hippocampus during and after acquisition of an ischemia-tolerant state. Brain Pathol 5:135–144

83.

Steller

(1995) Mechanisms and genes of cellular suicide. Science 267:1445–1449

84.

Tamara

Kirino

Sago

Takagi

Oka

(1990) Atrophy of ipsilateral substantia nigra following middle cerebral artery occlusion in the rat. Brain Res 510:154–157

85.

Torres-Munoz

Roberts

Wood

Nowak

Jr. Pulsinelli Petito

(1996) Delayed neuronal death in post-ischemic hippocampus is not associated with transcription or translation of the apoptotic genes p53 and interleukin converting enzyme-I (ICE). Neurosci 22:1179

86.

Vasilakos

Ghayur

Carroll

Giegel

Saunders

Quintal

Keane

Shivers

(1995) IL-1 beta converting enzyme (ICE) is not required for apoptosis induced by lymphokine deprivation in an IL-2 dependent T cell line. J Immunol 155:3433–3442

87.

Volpe

Wessel

Mukherjee

Federoff

(1995) Temporal pattern of internucleosomal DNA fragmentation in the striatum and hippocampus after transient forebrain ischemia. Neurosci Lett 186:157–160

88.

Wijsman

Jonker

Keijzer

Van deVelda

CJH

Cornelisse

Van Dierendonck

(1993) A new method to detect apoptosis in paraffin sections: In-situ end-labeling of fragmented DNA. J Histochem Cytochem 41:7–12

89.

Wyllie

Kerr

JFR

Currie

(1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306

90.

Wyllie

Morris

Smith

Dunlop

(1984) Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 142:67–77

91.

Yonish-Rovach

Resnitsky

Lotem

Sachs

Kimchi

Oren

(1991) Wild type p53 induces apoptosis of myeloid leukemic cells that is inhibited by interleukin-6. Nature 352:345–347

92.

Zakeri

Quaglino

Latham

Lockshin

(1993) Delayed internucleosomal DNA fragmentation in programmed cell death. FASEB J 7:470–478