Research reportEarly and sequential recruitment of apoptotic effectors after focal permanent ischemia in mice
Introduction
Biochemical and morphological characteristics of apoptotic cell death, such as cytoplasmic shrinkage, chromatin condensation and DNA fragmentation, have been detected in cerebral focal and global ischemia models in rodents (for review, see Ref. [2]). After focal ischemia in mice, the predominant localization of apoptotic cells at the inner boundary of the ischemic lesion suggests that the apoptotic process largely contributes to the expansion of the ischemic damage 7, 8, 18, 21. The apoptotic pathway is composed of at least three functional distinct phases: an initiation phase during which cells receive death stimuli; an effector phase dependent of Bcl-2 family members and of apoptogenic proteins released from mitochondria; and a degradation phase, dependent of caspases [16]. The Bcl-2 family proteins could function as channels regulating the mitochondrial permeability transition, and/or the release of apoptogenic proteins, such as the cytochrome c (also named Apaf-2) or Apoptosis Inducing Factor (AIF) 16, 29. The redistribution of cytochrome c from mitochondria to cytosol participates in in vitro neuronal apoptosis [25], by contributing to the proteolytic activation of caspase-9 and -3 19, 31. The cytochrome c is released in the cytosol after transient focal [5] and global [26] ischemia in rats. The activation and the cleavage of caspase-3 mediate delayed neuronal death after transient ischemia 1, 24. One of the best characterized substrate of caspase-3 is the poly(ADP–ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair and maintenance of genome integrity [27]. The cleavage of PARP by caspase-3 has been demonstrated in a model of transient global ischemia [1] and PARP knock-out mice exhibit a reduction of ischemic damage after a transient focal ischemia [4].
In a model of transient focal ischemia, the volume of infarction was significantly decreased in transgenic mice overexpressing the manganese superoxide dismutase (Mn SOD), an endogenous mitochondrial antioxidant [13] and mutant mice with Mn SOD deficiency presented an exacerbation of cerebral lesion [22]. Recently, it was shown that knock-out mice deficient in Mn SOD presented an enhanced release of cytochrome c as well as an enhanced DNA fragmentation after the occlusion of the middle cerebral artery (MCAO) in mice [6], suggesting a direct participation of Mn SOD in the apoptotic pathways elicited after ischemic shock.
We have analyzed different steps of ischemia-induced apoptosis, namely the subcellular distribution of cytochrome c, the expression of caspase-3, the cleavage of PARP and the DNA fragmentation in function of time intervals after MCAO in a model of focal permanent ischemia in mice. We report here that the kinetics of cytosolic increase of cytochrome c is in good concordance with the induction of caspase-3 expression in the infarcted area, and the delayed cleavage of PARP. Our result confirm the pivotal role of mitochondria as central executioner of the apoptotic machinery. But, we found no modifications of Mn SOD protein content or enzymatic activity indicating that Mn SOD does not seem recruited in this model.
Section snippets
Surgical procedure and localization of infarcted brain territory
All the studies with mice have been conducted in respect to the French and the EU legislations. Male (C57Bl/6×DBA/2)F1 mice (Charles River) weighing 20–25 g were anesthetized intraperitoneally with chloral hydrate (500 mg/kg). Coagulation of the left MCA was realized as previously described [8]. Under low power magnification, a skin incision was made vertically between the eye and the ear. The parotid gland and surrounding soft tissues were pushed downward and the underlying temporalis was
Description of the ischemic lesion
Following MCAO, the ischemic territory was strictly ipsi-lateral (left side) and localized exclusively in the temporo-parietal cortex (Fig. 1). The damaged area, visible as early as 30 min, expanded as a function of time interval to reach a maximum at 24 h [10]. Apoptotic cells were only present in the ischemic lesion and were detected as early as 1 h after MCAO in the boundary of ischemia territory [9]. Apoptotic cells were recognized either on the basis of morphological criteria after
Discussion
In this study, we report in a model of focal permanent ischemia in mice that the cytosolic increase of cytochrome c and the induction of caspase-3 immunoreactivity occurs exclusively in the damaged brain, and precedes the cleavage of PARP, a caspase-3 substrate. The kinetics of these events is in concordance with the later apparition of the internucleosomal fragmentation of DNA, the ultimate step of apoptosis. On the contrary, no differences of either Mn SOD protein content or Mn SOD activity
Acknowledgements
C.G. is supported by the Ministère de l'Education Nationale, de la Recherche, et de la Technologie. We thank Dr. Irène Ceballos-Picot (CNRS URA 1335, Hôpital Necker, Paris, France) for her help with Mn SOD enzymatic activity determination.
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