Broad-spectrum caspase inhibitors: from myth to reality?
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Amino Acid Chloromethyl Ketones↗
Biomedical subjects
C Charriaut-Marlangue
Publications and source records attributed to C Charriaut-Marlangue.
Glial activation in white matter following ischemia in the neonatal P7 rat brain.
This study examines cell death and proliferation in the white matter after neonatal stroke. In postnatal day 7 injured rat, there was a marked reduction in myelin basic protein (MBP) immunostaining mainly corresponding to numerous pyknotic immature oligodendrocytes and TUNEL-positive astrocytes in the ipsilateral external capsule. In contrast, a substantial restoration of MBP, as indicated by the MBP ratio of left-to-right, occurred in the cingulum at 48 (1.27 +/- 0.12) and 72 (1.30 +/- 0.18, P < 0.05) h of recovery as compared to age-matched controls (1.03 +/- 0.14). Ki-67 immunostaining revealed a first peak of newly generated cells in the dorsolateral hippocampal subventricular zone and cingulum at 72 h after reperfusion. Double immunofluorescence revealed that most of the Ki-67-positive cells were astrocytes at 48 h and NG2 pre-oligodendrocytes at 72 h of recovery. Microglia infiltration occurs over several days in the cingulum, and a huge quantity of macrophages reached the subcortical white matter where they engulfed immature oligodendrocytes. The overall results suggest that the persistent activation of microglia involves a chronic component of immunoinflammation, which overwhelms repair processes and contributes to cystic growth in the developing brain.
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Distribution of Aquaporin 9 in the adult rat brain: preferential expression in catecholaminergic neurons and in glial cells.
Aquaporin 9 (AQP9) is a recently cloned water channel that is permeable to monocarboxylate, glycerol and urea. In rat, AQP9 has been found in testis and liver as well as in brain where its expression has been initially shown in glial cells in forebrain. However, the expression of AQP9 has not been investigated in the brainstem. The purpose of this study is to describe the distribution of AQP9-immunoreactive cells throughout the adult rat brain using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. We performed immunolabeling on brain from animals perfused with fixative and we show that AQP9 is expressed (i) in astrocytes in the glia limitans, in the white matter and in glial cells of the cerebellum, (ii) in the endothelial cells of pial vessels, and (iii) in specific groups of neurons. The neuronal AQP9 expression was almost exclusively observed in catecholaminergic cells including the adrenergic, noradrenergic and dopaminergic groups, but not in other monoaminergic neurons such as serotonergic or histaminergic cells. A slight labeling was also observed in non-catecholaminergic neurons localized in the paraventricular nucleus of the hypothalamus. These results indicate that AQP9 has a unique brain distribution with a preferential localization in catecholaminergic nuclei known to be involved in many cerebral functions. While the presence of AQP9 in glia limitans and in endothelial cells of the pial vessels could be related to water transport through the blood-brain barrier, its expression in neuronal cells, not directly involved in the osmoregulation, suggests that brain AQP9 could also be used as a metabolite channel since lactate and glycerol can be energy substrates for neurons.
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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.
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[Neuronal death: potential role of the nuclear enzyme, poly (ADP-ribose) polymerase].
Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is known as a nuclear enzyme that is activated by DNA strand breaks to participate in DNA repair. It is also called poly(ADP-ribose) synthase (PARS) or poly(ADP-ribose) transferase (PADRT). In physiological conditions, PARP plays an important role in maintaining genomic stability. However, for several pathological situations, which include massive DNA injury (brain ischemia for example), excessive activation of PARP can deplete stores of nicotinamide adenine dinucleotide (NAD+), the PARP substrate, which, with the subsequent ATP depletion, leads to cell death. PARP activation appears to play a major role in neuronal death induced by cerebral ischemia, traumatic brain injury, Parkinson disease and other pathologies. PARP inhibitors (3-aminobenzamide and other compounds) and PARP gene deletion induced dramatic neuroprotection in experimental animals (rats, mice). Accordingly, these data suggest that PARP inhibitors could provide a novel therapeutic approach in a wide range of neurodegenerative disorders including cerebral ischemia and traumatic brain injury.
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Apoptosis in immature brain: a therapeutic approach to neuronal death.
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Journal Article↗
Contribution of NO/ONOO- pathway to the deleterious effect of traumatic brain injury in mice.
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Journal Article↗
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.
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DNA damage and DNA damage-inducible protein Gadd45 following ischemia in the P7 neonatal rat.
Cerebral ischemia in adult rodents leads to the production of several types of lesions in the genomic DNA, followed by the activation of the damage-response indicator Gadd45. Our purpose was to investigate the structural changes that occur in chromatin DNA and repair processes after ischemic injury in neonatal brain. Neonatal ischemia was induced by the permanent left MCA occlusion in association with 1 h occlusion of the left common carotid artery in 7-day-old Wistar pups. Oligonucleosome fragments that are recognized as the characteristic DNA ladder was observed in a delayed fashion. Double-strand breaks result in high molecular weight fragments of 50- and 300-kbp as demonstrated by pulsed-field gel electrophoresis, and visualized by the TUNEL assay at 24 h of recovery. In contrast, DNA single-strand breaks, shown by the use of DNA polymerase I-mediated biotin-dATP nick translation were not so abundant. Gadd45 immunoreactivity was sequentially increased in vulnerable neurons in the infarct (4 to 24 h) and in sublethally injured neurons in the penumbra (24-48 h). Taken together, these findings suggest that Gadd45 responds to DNA damage following neonatal ischemia. Furthermore, repairing processes seem to be more active in the penumbra and therefore Gadd45 could have also a protective role in cerebral ischemia.
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Pentylenetetrazol seizures induce cell suffering but not death in the immature rat brain.
To investigate whether long-term functional consequences of status epilepticus (SE) induced by pentylenetetrazol in 10-day-old rats correlated with cell injury and/or death, acid fuchsin and TUNEL staining were performed between 4 to 144 h after SE. Acid fuchsin stained hippocampus, amygdala and cerebral cortex at 24 h but not at 72 and 144 h. No DNA fragmentation was apparent at any time. Thus, immature neurons subjected to sustained seizures suffer transiently but survive probably by activating repair processes.
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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.
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[Apoptosis and cerebral ischemia].
Although physiological cell death has been known for decades, interest in the subject was renewed in 1972 when Kerr, Wyllie and Currie described in detail the ultrastructural changes characteristic of dying cells and coined the term apoptosis to describe the process. When dying during development or following a toxic insult, cells display a wide variety of morphological changes. A binary classification scheme suggests that physiologically appropriate death is due to apoptosis, and that pathological mechanisms involve necrosis. In this report, we will address developments in our understanding of a potential involvement of apoptotic cell death in ischemia which induce selective and delayed neuronal degeneration. Such results may open important steps in therapeutic approaches for the preservation of neurons.
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Reduction of tyrosine nitration after N(omega)-nitro-L-arginine-methylester treatment of mice with traumatic brain injury.
Oxygen free radicals and nitric oxide (NO) have been proposed to be involved in the cascade of injury elicited by traumatic brain injury. However, the mechanism(s) of injury remain to be explored. Since superoxide generation is triggered by traumatic brain injury, the cytotoxic peroxynitrite could be formed, but it is not known if this actually occurs. Dot blot and immunohistochemistry studies were performed to quantify tyrosine nitration and identify cell types in which such reactions occur in the brain of mice submitted to traumatic brain injury. Nitrotyrosine formation increased from 4 to 24 h after traumatic brain injury and was primarily observed in degenerating neurons, in areas corresponding to the sites of direct impact (frontal cortex) and diffuse impact (frontoparietal cortex and ventromedial hypothalamic nucleus). Furthermore, N omega-nitro-L-arginine-methylester (L-NAME), a NO-synthase inhibitor which has previously been shown to promote neurological recovery in traumatic brain injury, reduced nitrotyrosine formation and the number of nitrotyrosine-positive neurons. These results indicate that traumatic brain injury induces peroxynitrite formation which may contribute to cell damage.
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A gliotoxic factor and multiple sclerosis.
The pathogenesis of multiple sclerosis (MS) is unknown. Searching for possible toxic factors, it was found that 3-day exposure to heat-treated cerebrospinal fluid (CSF) from MS patients caused apoptotic death of astrocytes and oligodendrocytes, but not fibroblasts, myoblasts, Schwann cells, endothelial cells and neurons, in vitro. CSFs from other inflammatory or non-inflammatory neurological diseases showed no toxicity. Exposure of these glial cells to partially purified MS CSF produced DNA fragmentation, apoptotic bodies, chromatin condensation, cell shrinkage, and changes in the levels of known cytokines. A cytotoxic factor, called gliotoxin, was characterized chromatographically as a stable 17-kDa glycoprotein. Since this protein is highly cytotoxic for astrocytes and oligodendrocytes, it may represent an initial pathogenic factor, leading to the neuropathological features of MS, such as blood-brain barrier involvement and demyelination.
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Apoptosis and programmed cell death: a role in cerebral ischemia.
Hypoxic-ischemic neuronal death has long been considered to represent necrosis, but it now appears that many brain neurons undergo apoptosis after either global or focal ischemic insults. Recent studies demonstrated: 1) DNA cleavage into oligonucleosome-sized fragments demonstrated by a typical ladder pattern; 2) early endonuclease activation, as demonstrated by the presence of high molecular weight DNA fragments (300 to 50 kbp); 3) chromatin condensation and apoptotic bodies formation; 4) activation of apoptosis-associated proteins. These results may indicate that apoptosis contributes to the development of the ischemic infarct and is probably substantially distinct from ischemia-triggered excitotoxicity, which tends to produce necrosis.
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Ultrastructural morphology of neuronal death following reversible focal ischemia in the rat.
Electron microscopy and terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labelling (TUNEL) were used to illustrate the different stages and subcellular alterations of cell degeneration that occur in the striatum of the rat after transient focal ischemia. Degenerating neurons exhibited different morphological types: apoptosis Type 1 (aggregation of dense masses of chromatin beneath the 'intact' nuclear membrane) and Type 2 (high cytoplasmic vacuolization), and necrosis. These profiles were localized in different part of the striatum. Type 1 was found in the head of the caudate putamen, Type 2 in the middle part of the striatum and necrosis in the striatal core. These ultrastructural results demonstrated that apoptosis occurs in neurons following focal ischemia in the striatal penumbra. In contrast, necrosis can be observed in the ischemic core, the region maximally affected by the ischemia. Finally, the presence of astrocytes throughout both the penumbra and ischemic core displaying numerous cytoplasmic vacuoles suggested an activation of glial cells.
Journal Article↗
Nitric oxide production and perivascular tyrosine nitration following focal ischemia in neonatal rat.
Oxygen free radicals and nitric oxide (NO.) have been proposed to be involved in acute CNS injury produced by cerebral ischemia; however, controversy remains regarding how they cause injury. Because superoxide generation is triggered during reperfusion, the cytotoxic oxidant peroxynitrite could be formed, but it is not known if this occurs. Dot blot and immunohistochemistry studies were performed on the magnitude and time course of tyrosine nitration and inducible NO synthase (NOS2) in the postischemic rat pup brain. Neonatal ischemia was induced by permanent left middle cerebral artery occlusion in association with 1-h occlusion of the left common carotid artery in 7-day-old Wistar pups. Nitrotyrosine (NT) immunoreactivity was evident in the blood vessels close to the cortical infarct at 48-72 h of recovery, and T lymphocytes were involved with this production. NOS2 immunoreactivity was seen in neutrophils in the same vessels and in the parenchyma at 72 h of recirculation. Whereas NT staining decreased with time, NOS2-positive neutrophils could be still detected in arachnoid vessels at 14 days of recirculation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events that lead to brain oxidative stress in neonatal ischemia. Moreover, NO-related species may serve as a signaling function instead of directly mediating toxicity.
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A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: morphological changes indicative of apoptosis.
BACKGROUND AND PURPOSE: The mechanisms leading to delayed cell death after hypoxic-ischemic injury in the developing brain remain to be elucidated. The aim of this study was to develop a model of transient focal ischemia in the neonatal rat in an attempt to create a reperfusion phase since in the filament model of reversible middle cerebral artery occlusion, size limitations precluded performing this procedure before 14 to 18 days. We then analyze whether apoptosis or necrosis occurs in this model. METHODS: Seven-day-old Wistar rat pups (n = 96) underwent permanent left middle cerebral artery occlusion in association with 1-hour occlusion of the left common carotid artery. Evolution of the brain infarction was studied from 24 hours to 3 months on cresyl violet-stained coronal sections. Infarct volume was determined with the use of the mitochondrial stain 2,3,5-triphenyltetrazolium chloride. Neuronal death was demonstrated by the silver staining method of Gallyas et al (1980). Chromatin condensation was shown by DNA fragmentation assessed with the use of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay in cryostat sections and electron microscopic analysis. RESULTS: Almost all of the animals who survived had reproducible cortical infarcts. The mean infarct volume was 31+/-7 mm3 (mean+/-SD). The ipsilateral hemisphere showed a well-delineated lesion in the frontoparietal cortex at 3-month recovery. Argyrophilic (dying) neurons were observed a few hours after reperfusion and increased with time. Cells exhibiting DNA fragmentation were shown as early as 6 hours, increased up to and peaked at 24 to 96 hours, then progressively decreased and persisted for several days, suggesting an ongoing process. Electron microscopy analysis demonstrated high condensation and clumping of chromatin beneath nuclear membrane in shrunken neurons. CONCLUSIONS: 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.
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