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Hypoxia-ischaemia in the developing brain results in brain injury with prominent features of apoptosis. In the present study, a third generation dipeptidyl broad-spectrum caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VDOPh), was tested in a model of unilateral focal ischaemia with reperfusion in 7-day-old rats. Q-VD-OPh (1 mg/kg, i.p.) reduced cell death, resulting in significant neuroprotection at 48 h of recovery (infarct volume of 12.6 ± 2.8 vs. 24.3 ± 2.2%, p ¼ 0.006). The neuroprotective effects observed at 48 h post-ischaemia hold up at 21 days of survival time and attenuate neurological dysfunction. Analysis by gender revealed that females were strongly protected (6.7 ± 3.3%, p ¼ 0.006), in contrast to males in which there was no significant effect, when Q-VD-OPh was given after clip removal on the left common carotid artery. Immunoblot analysis demonstrated that Q-VD-OPh inhibits caspase 3 cleavage into its p17 active form and caspase 1 up-regulation and cleavage in vivo. Following ischaemia in P7 rats, males and females displayed different time course and pattern of cytochrome c release and active p17 caspase 3 during the first 24 h of recovery. In contrast, no significant difference was observed for caspase 1 expression between genders. These results indicate that ischaemia activates caspases shortly after reperfusion and that the sex of the animal may strongly influences apoptotic pathways in the pathogenesis of neonatal brain injury. The specificity, effectiveness, and reduced toxicity of Q-VD-OPh may determine the potential use of peptide-derived irreversible caspase inhibitors as promising therapeutics.

mentioned that embryonic bodies cultured in the presence of rotenone were smaller than those lacking AIF, suggesting subtle differences between the acute complex I defect induced by rotenone (that might affect homeostatic systems in the cell other than respiration) and the chronic defect induced by the AIF knockout.These results illustrate the difficulty to separate the two functions of AIF, first as a mitochondrial redox enzyme required for normal respiratory function and, second, as a factor that can participate in the apoptotic execution phase, after its translocation to the nucleus. The observation that AIF participates in cavitation-associated apoptosis 9 implicitly suggested that cavitation would depend on the lethal action of AIF. Based on recent insights on AIF biology, however, it is possible that at least some of the lethal processes that were interrupted by the removal of AIF from the experimental system actually were suppressed due to defective bioenergetic and/or redox metabolism. As a result, we recommend the use of rotenone as an internal control in experiments, in which the apoptosis-modulatory effects of human or mouse AIF are assessed. Only when rotenone fails to modify the apoptotic response, the interpretation that the lethal (rather than the vital) action of AIF is involved in the process can be maintained.Knock-in mutations that affect only the lethal function of AIF yet do not interfere with its metabolic activity in mitochondria are being prepared in several laboratories, 2 and the use of such mutants will yield important insights into the contribution of AIF to lethal signal transduction processes. Such knock-in mutations will also help to decipher the contribution of AIF, alone or in combination with mutations of the apoptosome components, to programmed cell death in physiological and pathological cell death.

Our study demonstrates the feasibility of performing ischemia-reperfusion in 7-day-old rats that develop progressive neuronal death with features characteristic of apoptosis. The reperfusion phase mimics events that occur during neonatal human hypoxic-ischemic encephalopathy at birth, since perinatal intensive care most often permits recirculation.

Apoptosis is one of the two forms of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alternations including membrane blebbing and nuclear and cytoplasmic condensation. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385-388). We now show that a significant number of striatal and cortical neurons, exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, were also shown at 6 h recirculation, in the ischemic core. Further investigation with hematoxylin/eosin staining revealed that apoptotic nuclei were present in cells with a large and swelled cytoplasm and in cells with an apparently well-preserved cytoplasm. These two types of cell death were reminiscent of those described in developmental cell death. Our data suggested that apoptosis may contribute to the expansion of the ischemic lesion.

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Background and Purpose-The contribution of inflammatory response to the pathogenesis of ischemic lesions in the neonate is still uncertain. This study described the chronological sequence of inflammatory changes that follow cerebral ischemia with reperfusion in the neonatal P7 rat. Methods-P7 rats underwent left middle cerebral artery electrocoagulation associated with 1-hour left common carotid artery occlusion. The spatiotemporal pattern of cellular responses was characterized immunocytochemically with the use of antibodies against rat endogenous immunoglobulins to visualize the area of the breakdown of the blood-brain barrier.Infiltration of neutrophils and T lymphocytes was demonstrated by antibodies against myeloperoxidase and a pan-T cell marker, respectively. Antibodies ED1 and OX-42 were applied to identify microglial cells and macrophages. The response of astrocytes was shown with antibodies against glial fibrillary acidic protein. Cell survival was assessed by Bcl-2 expression. Cell death was demonstrated by DNA fragmentation with the use of the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) assay and Bax immunodetection. Results-Endogenous immunoglobulin extravasation through the blood-brain barrier occurred at 2 hours of recirculation and persisted until 1 month after ischemia. Neutrophil infiltration began at 24 hours and peaked at 72 to 96 hours (30Ϯ3.4 neutrophils per 0.3 mm 2 ; PϽ0.0001), then disappeared at 14 days after ischemia. T cells were observed between 24 and 96 hours of reperfusion. Resident microglia-macrophages exhibited morphological remnants and expressed the cell death inhibitor Bcl-2 at 24 hours of recirculation. They became numerous within the next 48 hours and peaked at 7 days after ischemia. Phenotypic changes of resident astrocytes were apparent at 24 hours, and they proliferated between 48 hours and 7 days after ischemia. Progressively inflammatory cells showed DNA fragmentation and the cell death activator Bax expression. Cell elimination continued until there was a complete disappearance of the frontoparietal cortex.Conclusions-These data demonstrate that perinatal ischemia with reperfusion triggers acute inflammatory responses with granulocytic cell infiltration, which may be involved in accelerating the destructive processes. (Stroke. 1999;30:1916-1924.)