Novel Apoptotic Evidence for Delayed Neuronal Death in the Hippocampal CA1 Pyramidal Cells After Transient Ischemia
To the Editor:
We read with great interest the article by Colbourne et al1 recently published in Stroke. The authors investigated the degree and maturation rate of hippocampal CA1 neuronal damage as a function of the duration of ischemia. They concluded that brief forebrain ischemia results in a slower progression of CA1 loss than do more severe insults. Furthermore, they reported that some neuroprotective agents, NBQX and SNX-111, influenced the maturation rate but only postponed the neuronal damage. While we agree with these results, we concur with the concerns expressed in the Editorial Comment on this article regarding their conclusions that the delayed neuronal death (DND) is not apoptosis, but is in fact necrosis. This conclusion was made on the basis of the lack of the morphological evidence for apoptosis by ultrastructural examination. However, morphological change during apoptosis occurs dynamically, with initial steps occurring within the first few hours. It is, therefore, very important to observe the early phase of apoptotic process to obtain the morphological evidence for apoptosis.
The authors performed the ultrastructural study 7 days after 15 minutes of ischemic insult. We think that this time point is too long after the initiating insult. In their time-course data using light microscopic examination, 74% of CA1 neurons subjected to 15 minutes of ischemia exhibited damage as early as 3 days after ischemia. It is speculated that electron microscopic evidence of apoptosis has disappeared by 7 days after the ischemia. In fact, an electron microscopic study by Nitatori et al,2 which was performed in accordance with the original method of DND,3 demonstrated the apoptotic evidences at 3 days after ischemia.
Morphological characteristics themselves do not provide an unambiguous definition of apoptosis. Other studies, such as DNA fluorescence assay, in situ terminal deoxynucleotidyl transferase–mediated dUTP nick-end-labeling (TUNEL) method,4 gel electrophoresis,5 and caspase activation,6 indicate that DND is a form of programmed cell death, or apoptosis. It remains unknown, however, whether DND is necrosis or apoptosis.
Recently, we reported a sequential TUNEL technique as a method for tract tracing to demonstrate novel apoptotic evidence for DND in hippocampal CA1 pyramidal cells after transient ischemia.7 In this study, we demonstrated the migration of the fragmented DNA from the nuclei into the apical dendrite in the CA1 pyramidal cells after ischemia. The migration of the fragmented DNA into apical dendrites in stratum radiatum of the hippocampus was first observed 66 hours after the ischemic insult. The fragmented DNA localized in the dendrites appeared most prominently at 96 hours (see arrowheads in the Figure⇓). Pooling of the fragmented DNA around the dendrite terminal end in the stratum lacunosum moleculare of the hippocampus was observed at this time (see arrows in the Figure⇓). The localization of fragmented DNA in apical dendrites was confirmed by DNA fluorescence staining.8
The characteristic morphological changes such as chromatin condensation, its movement to the nuclear periphery, and apoptotic body formation in the typical apoptotic cells require ATP-dependent energy support. Interestingly, ATP is essential for morphological changes occurring in nuclei during apoptosis but not for DNA fragmentation.9 Axonal or dendritic transport also require ATP-dependent energy support. Yasumoto et al10 have reported that the ATP levels in the stratum radiatum were 118% and 43% of those of control at 2 and 4 days after ischemia, respectively. It is speculated that the dendritic DNA flow based on ATP metabolism is over within 4 days.
Necrosis is defined, in other words, as a failure of cellular metabolism. Our results reveal ATP-dependent transport system of CA1 pyramidal neurons after transient ischemia is still active after nuclear DNA fragmentation, which is considered to be a late event in the apoptotic process. This finding strongly supports the hypothesis that DND is indeed apoptotic cell death. We also wish to emphasize that the dynamic events of apoptotic process of DND are transient and are complete within 4 days.
- Copyright © 2000 by American Heart Association
Colbourne F, Li H, Buchan AM, Clemens JA. Continuing postischemic neuronal death in CA1: influence of ischemia duration and cytoprotective doses of NBQX and SNX-111 in rats. Stroke. 1999;30:662–668.
Nitatori T, Sato N, Waguri S, Karasawa Y, Araki H, Shibanai K, Kominami E, Uchiyama Y. Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J Neurosci. 1995;15:1001–1011.
Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 1982;239:57–69.
Hara A, Niwa M, Nakashima M, Iwai T, Uematsu T, Yoshimi N, Mori H. Protective effect of apoptosis-inhibitory agent, N-tosyl-l-phenylalanyl chloromethyl ketone against ischemia-induced hippocampal neuronal damage. J Cereb Blood Flow Metab. 1998;18:819–823.
Heron A, Pollard H, Dessi F, Moreau J, Lasbennes F, Ben-Ari Y, Charriaut-Marlangue C. Regional variability in DNA fragmentation after global ischemia evidenced by combined histological and gel electrophoresis observations in the rat brain. J Neurochem. 1993;61:1973–1976.
Chen J, Nagayama T, Jin K, Stetler RA, Zhu RL, Graham SH, Simon RP. Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J Neurosci. 1998;18:4914–4928.
Hara A, Niwa M, Iwai T, Nakashima M, Yano H, Uematsu T, Yoshimi N, Mori H. Transport of fragmented DNA in apical dendrites of gerbil CA1 pyramidal neurons following transient forebrain ischemia. Brain Res. 1998;806:274–277.
Hara A, Niwa M, Iwai T, Nakashima M, Bunai Y, Uematsu T, Yoshimi N, Mori H. Neuronal apoptosis studied by a sequential TUNEL technique: a method for tract-tracing. Brain Res Protoc. 1999;4:140–146.
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Our studyR1 examined the light and ultrastructural characteristics of CA1 neuronal death following 5 minutes of ischemia (14-day survival) or 15 minutes of ischemia (7-day survival). We did not find any morphological evidence classically described as apoptosis (eg, apoptotic bodies). It is possible that we simply missed these features, which may have occurred earlier. However, we were specifically interested in comparing two different rates of DND in CA1, which certainly did occur at those times sampled. Furthermore, in a comparable studyR2 , in which CA1 DND was examined at 4, 14, and 60 days in gerbils subjected to ischemia with and without delayed hypothermic neuroprotection, we still did not observe any morphological signs of apoptosis. Again, CA1 neuronal death occurred throughout this period, because some CA1 loss was postponed by the delayed hypothermia treatment. Many others have similarly failed to see morphological evidence for apoptosis (eg, References 3 through 5) at these and earlier survival times.
It is perhaps true that morphological findings cannot rule out biochemical events such as occur with programmed cell death. Indeed, as stated in our articleR1 and as noted in both the editorial comment by ClemensR6 and the above letter by Hara et al, there are several events that occur in CA1 neurons destined to die which also occur in programmed cell death (eg, caspase activation). However, it has yet to be clearly proved whether these events are causal or merely coincidental with CA1 DND. Furthermore, some of this evidence, such as TUNEL staining, is nonspecific and occurs with a necrotic-type cell death.R7 Other evidence, such as use of caspase inhibitors to reduce CA1 loss, is also suspect, because the observations have not always been replicatedR8 and, importantly (as we made clear in our study), the survival times have been inadequate (eg, 4-day survival timeR9 ). Other evidence supporting apoptosis in ischemia, such as laddered DNA fragmentation, is now known to be atypical of classic apoptosis.R10
Regardless of the “mode” of CA1 neuronal death, we feel that, until now, specific antiapoptotic strategies and indeed other pharmacological treatments (eg, NBQX and SNX-111) have been very disappointing in the treatment of global ischemic injury, especially when compared with the indefatigable neuroprotection afforded by delayed postischemic hypothermia.R2 R11 R12 While hypothermia may affect events of an apoptotic nature, we believe that the multitude of its protective effects is what make hypothermia so potent. Accordingly, combination therapies aimed at treating most or all of the significant metabolic derangements (whatever they may be associated with), and not just “antiapoptotic” drugs, are the most fruitful way to proceed. Ischemic neuronal death must be studied “in its own right”R13 and not forced into an artificial mold (eg, necrosis versus apoptosis) for convenience.
Colbourne F, Li H, Buchan AM. Continuing postischemic neuronal death in CA1: influence of ischemia duration, and cytoprotective doses of NBQX and SNX-111. Stroke.. 1999;30:662–668.
Colbourne F, Sutherland GR, Auer RN. Electron microscopic evidence against apoptosis as the mechanism of neuronal death in global ischemia. J Neurosci.. 1999;19:4200–4211.
Deshpande J, Bergstedt K, Lindén T, Kalimo H, Wieloch T. Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death. Exp Brain Res.. 1992;88:91–105.
Petito CK, Torres-Munoz J, Roberts B, Olarte J-P, Nowak TS, Pulsinelli WA. DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischemia in the rat. J Cereb Blood Flow Metab.. 1997;17:967–976.
Petito CK, Olarte J-P, Roberts B, Nowak TS Jr, Pulsinelli WA. Selective glial vulnerability following transient global ischemia in rat brain. J Neuropathol Exp Neurol.. 1998;57:231–238.
Clemens JA. Editorial comment on “Continuing Postischemic Neuronal Death in CA1: Influence of Ischemia Duration and Cytoprotective Doses of NBQX and SNX-111 in Rats.” Stroke.. 1999;30:667–668.
Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte-Hermann R. In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology.. 1995;21:1465–1468.
Li H, Colbourne F, Sun P, Zhao Z, Buchan AM. Caspase inhibitors reduce neuronal injury following focal but not global cerebral ischemia in rats. Stroke. In press.
Hara A, Niwa M, Nakashima M, Iwai T, Uematsu T, Yoshimi N, Mori H. Protective effect of apoptosis-inhibitory agent, N-tosyl-l-phenylalanyl chloromethyl ketone against ischemia-induced hippocampal neuronal damage. J Cereb Blood Flow Metab.. 1998;18:819–823.
MacManus JP, Fliss H, Preston E, Rasquinha I, Tuor U. Cerebral ischemia produces laddered DNA fragments distinct from cardiac ischemia and archetypal apoptosis. J Cereb Blood Flow Metab.. 1999;19:502–510.
Colbourne F, Corbett D. Delayed postischemic hypothermia: a six month survival study using behavioral and histological assessments of neuroprotection. J Neurosci.. 1995;15:7250–7260.
Colbourne F, Li H, Buchan AM. Indefatigable CA1 sector neuroprotection with mild hypothermia induced 6 hours after severe forebrain ischemia in rats. J Cereb Blood Flow Metab.. 1999;19:742–749.
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