Apoptotic mitochondrial pathway in neurones and astrocytes after neonatal hypoxia-ischaemia in the rat brain.

Neuronal apoptosis plays an essential role in early brain development and contributes to secondary neuronal loss after acute ischaemia. Recent studies have provided evidence that caspase-3 is an important downstream event after hypoxia-ischaemia in the immature brain, but a minor event in the adult brain. Our investigations have focused on cell populations that expressed apoptotic effectors in the enzymatic death pathway including cytochrome c, caspase-9 and caspase-3. Expression, activation and cellular localization of these proteins were studied using cleavage of fluorogenic substrate and immunohistochemistry in neonatal rat brain after unilateral focal ischaemia. Caspase-3 enzyme activity was elevated in brain homogenate between 6 and 48 h after reperfusion. This activation was preceded by that of caspase-9, between 3 and 24 h. Apoptotic cell death was finally accomplished by poly-ADP-ribose polymerase cleavage, an endogenous caspase-3 substrate. In addition, immunodetection demonstrated that cytochrome c and activated caspase-9 and caspase-3 were expressed not only in the neurones, the primarily affected cells, but also within the astrocytes, which constituted a dense network delineating the infarct. These results suggested that glial injury may promote the formation of cystic lesions such as those observed clinically in the newborn brain.

[Cystic lymphangiomas of the mesentery and the epiploon. About two cases]. / Le lymphangiome kystique du mésentère et de l'épiploon. A propos de deux cas.

Two cases of cystic lymphangiomas of the mesentery and the epiploon are hereby related. The authors underline the following points: first, the rarity of these tumors along with the absence of significant clinical features makes the differential diagnosis with ovary tumors a difficult task. Second, although abdominal ultra sonography, completed by CT scan, allows the hypothesis of lymphangioma, only histological examination will confirm the diagnosis. There is no other treatment but complete exeresis, with a good prognosis and exceptional recurrences.

Poly(ADP-ribose) synthase inhibition reduces ischemic injury and inflammation in neonatal rat brain.

Poly(ADP-ribose) synthase (PARS), an abundant nuclear protein, has been described as an important candidate for mediation of neurotoxicity by nitric oxide. However, in cerebral ischemia, excessive PARS activation may lead to energy depletion and exacerbation of neuronal damage. We examined the effect of inhibiting PARS on the (a) degree of cerebral injury, (b) process of inflammatory responses, and (c) functional outcomes in a neonatal rat model of focal ischemia. We demonstrate that administration of 3-aminobenzamide, a PARS inhibitor, leads to a significant reduction of infarct volume: 63 +/- 2 (untreated) versus 28 +/- 4 mm(3) (treated). The neuroprotective effects currently observed 48 h postischemia hold up at 7 and 17 days of survival time and attenuate neurological dysfunction. Inhibition of PARS activity, demonstrated by a reduction in poly(ADP-ribose) polymer formation, also reduces neutrophil recruitment and levels of nitrotyrosine, an indicator of peroxynitrite generation. Taken together, our results demonstrate that PARS inhibition reduces ischemic damage and local inflammation associated with reperfusion and may be of interest for the treatment of neonatal stroke.

Inflammatory responses in the cerebral cortex after ischemia in the P7 neonatal Rat.

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 (30+/-3.4 neutrophils per 0.3 mm(2); P<0.0001), 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.

Case report: DNA fragmentation in glial cells in a cerebral biopsy from a multiple sclerosis patient.

Multiple sclerosis is characterized by myelin destruction and oligodendrocyte loss. The neuropathological hallmark of the disease is the presence of demyelinated plaques in the central nervous system. We have recently found a gliotoxic factor in MS cerebrospinal fluid which induces programmed cell death in vitro, in glial cells. Here we show DNA fragmentation and glial cell death in biopsy samples, obtained from a patient who underwent surgery with suspicion of tumor, and whose disease record, including brain autopsy, demonstrated an active multiple sclerosis. We used the in situ TUNEL technique, a method which sensitively detects the DNA fragmentation accompanying programmed cell death in tissue sections, and compatible with classical fixation techniques. We found intense DNA fragmentation in nuclei of glial cells at-or very near-to the site of demyelination. A double labeling technique showed that glial fibrillary associated protein positive astrocytes may undergo programmed cell death in multiple sclerosis.

Regulation of apoptosis-associated proteins in cell death following transient focal ischemia in rat pups.

Apoptosis is a process whereby developmental or environmental stimuli activate a genetic programme to execute a specific series of events that culminate in the death and efficient disposal of a cell. Although a series of recent data suggested that neuronal death following cerebral ischemia occurs through an apoptotic pathway, additional work is needed to establish the existence of a causal relationship between gene expression and DNA breaks in neuronal death. We investigate the role of p53 and Bax proteins in the induction of apoptosis induced by a new transient focal ischemia model in the rat pup. Our results show that wild-type p53 exerts a significant and time-dependent effect in the initiation of apoptosis, and that apoptosis is induced via DNA-strand breakage. Subsequently, increased Bax expression was observed in the cytoplasm of dying cells located in the infarct, whereas an increased Bcl-2 and hsp72 staining was detectable in survival cells and reactive glia present at the periphery of the lesion.

[Gliotoxic factor and multiple sclerosis]. / Un facteur gliotoxique et la sclérose en plaques.

Multiple sclerosis in a disease of the central nervous system characterized by perivascular and periventricular lesions of the myelin and immune cell infiltrates and increased permeability of the blood-brain barrier. We have found a cytotoxic factor of the cerebrospinal fluid (CSF) specific for multiple sclerosis patients which has 2 main characteristic effects in vitro on primary or immortalized astrocyte cultures: (1) disruption of the gliofilament network of the cells; and (2) apoptotic cell death induction. Moreover, in vivo, intraventricular injections of minute amounts of partially purified gliotoxic factor in adult rats have striking effects on both the morphology and general organization of astrocytes in the entire brain and the permeability characteristics of the blood brain barrier, which becomes leaky to immunoglobulins. These pathological effects are strongly similar to some of the neuropathological findings reported during the course of MS--They suggest an entirely new hypothesis to explain the active stage of the disease: the presence of a new factor of unknown extrinsic (viral) or intrinsic (cellular) origin, able to disorganize the glial cytoskeleton and glial cell differentiation. This factor is then able to provoke glial cell death. Such glial cell death may result in both demyelination and increased blood brain barrier permeability. Both in vitro and in vivo studies strongly support the idea that this gliotoxic factor plays a central role in the pathogenesis of MS, making its full identification a critical theme for MS research.