Sylvain Renolleau

Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender.

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‐VD‐OPh), 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 = 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 = 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.

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.

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.

Gender-Related Differences in Apoptotic Pathways After Neonatal Cerebral Ischemia:

Many central nervous system (CNS) diseases display sexual dimorphism, specifically a predilection for one gender or a gender-dependent response to treatment. Exposure to circulating sex steroids is felt to be a chief contributor to this phenomenon. However, CNS diseases of childhood and of the elderly also demonstrate gender predominance and/or sexual dimorphism response to therapies. In this short update, we provide information concerning one of the most interesting new emerging concepts related to the influence of the sex in the pathogenesis of developmental brain injuries leading to different levels of neuroprotection between genders after cerebral hypoxia-ischemia or ischemia. NEUROSCIENTIST 14(1):46—52, 2008. DOI: 10.1177/1073858407308889

Nitric oxide production and perivascular tyrosine nitration following focal ischemia in neonatal rat

Abstract: 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.

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.

Ventilatory responses to hypercapnia and hypoxia in Mash-1 heterozygous newborn and adult mice.

Normal control of breathing is characterized by maintenance of CO2 and O2 arterial pressures at constant levels by appropriate ventilatory responses to changes in CO2 production and O2 consumption. Abnormal development of this regulatory system during embryogenesis may produce early impairments in chemosensitivity, as in congenital central hypoventilation syndrome. The present study addresses the role of the mammalian achaete-scute homologous gene (Mash-1) in the development of respiratory control. We analyzed ventilatory responses to hypercapnia (8% CO2, 21% O2, 71% N2) and hypoxia (10% O2, 3% CO2, 87% N2) in newborn and adult Mash-1 heterozygous mice (Mash-1+/−) and their wild-type littermates (Mash-1+/+). Ventilation, breath duration, and tidal volume were measured using whole-body plethysmography. Ventilatory responses to hypercapnia were significantly weaker in newborn male Mash-1+/− compared with Mash-1+/+ mice as a result of a weaker breath-duration response. No differences were observed between adult Mash-1+/− and Mash-1+/+ mice. Our data suggest that Mash-1 may be involved in respiratory control development via mechanisms linked to the X chromosome.

Melatonin Promotes Myelination by Decreasing White Matter Inflammation After Neonatal Stroke

Melatonin demonstrates neuroprotective properties in adult models of cerebral ischemia, acting as a potent antioxidant and anti-inflammatory agent. We investigated the effect of melatonin in a 7-d-old rat model of ischemia-reperfusion, leading to both cortical infarct and injury in the underlying white matter observed using MRI and immunohistochemistry. Melatonin was given i.p. as either a single dose before ischemia or a double-dose regimen, combining one before ischemia and one 24 h after reperfusion. At 48 h after injury, neither a significant reduction in cortical infarct volume nor a variation in the number of TUNEL- and nitrotyrosine-positive cells within the ipsilateral lesion was observed in melatonin-treated animals compared with controls. However, a decrease in the density of tomato lectin-positive cells after melatonin treatment was found in the white matter underlying cortical lesion. Furthermore, we showed a marked increase in the myelin basic protein-immunoreactivity in the cingulum and in the density of mature oligodendrocytes (APC-immunoreactive) in both the ipsilateral cingulum and external capsule. These results suggest that melatonin is not able to reduce cortical infarct volume in a neonatal stroke model but strongly reduces inflammation and promotes subsequent myelination in the white matter.

Inhaled Nitric Oxide Reduces Brain Damage by Collateral Recruitment in a Neonatal Stroke Model

Background and Purpose— We recently demonstrated that endogenous nitric oxide (NO) modulates collateral blood flow in a neonatal stroke model in rats. The inhalation of NO (iNO) has been found to be neuroprotective after ischemic brain damage in adults. Our objective was to examine whether iNO could modify cerebral blood flow during ischemia–reperfusion and reduce lesions in the developing brain. Methods— In vivo variations in cortical NO concentrations occurring after 20-ppm iNO exposure were analyzed using the voltammetric method in P7 rat pups. Inhaled NO-mediated blood flow velocities were measured by ultrasound imaging with sequential Doppler recordings in both internal carotid arteries and the basilar trunk under basal conditions and in a neonatal model of ischemia–reperfusion. The hemodynamic effects of iNO (5 to 80 ppm) were correlated with brain injury 48 hours after reperfusion. Results— Inhaled NO (20 ppm) significantly increased NO concentrations in the P7 rat cortex and compensated for the blockade of endogenous NO synthesis under normal conditions. Inhaled NO (20 ppm) during ischemia increased blood flow velocities and significantly reduced lesion volumes by 43% and cellular damage. In contrast, both 80 ppm iNO given during ischemia and 5 or 20 ppm iNO given 30 minutes after reperfusion were detrimental. Conclusions— Our findings strongly indicate that, with the appropriate timing, 20 ppm iNO can be transported into the P7 rat brain and mediated blood flow redistribution during ischemia leading to reduced infarct volume and cell injury.

Severe respiratory syncytial virus bronchiolitis in children: from short mechanical ventilation to extracorporeal membrane oxygenation

The objective of this study was to describe the characteristics of children who required mechanical ventilation (MV) or extracorporeal membrane oxygenation (ECMO) support for respiratory syncytial virus (RSV) bronchiolitis, and to identify risk factors associated with disease severity assessed by duration of MV, mortality and need for ECMO. Ventilated children under 1 year of age admitted for bronchiolitis were retrospectively studied over the 8-year period 1996–2003. The study population included 151 children. Of these, 38.4% were born prematurely and 8.6% had bronchopulmonary dysplasia (BPD). The mean age at initiation of MV was 61 days (±63 days). Infants were ventilated for a mean of 7.8 days (±7.5 days). Multivariate analysis revealed that prolonged duration of MV (>6 days, median value) was significantly associated with low gestational age ( P =0.02 for the group <32 weeks), requirement of neonatal oxygen supplementation ( P =0.03), BPD ( P =0.02) and positive tracheal aspiration culture ( P =0.004), in particular for Haemophilus influenzae ( P =0.03). Fourteen infants required ECMO with a mean period of MV before ECMO of 3.9 days (±4.5 days). Amongst these infants, the frequency of BPD was significantly higher as compared with the others ( P =0.001). Four infants died (survival rate 71.4%). The mean duration of ECMO for survivors was 12.1 days (±3.3 days). Conclusion:The data suggest that gestational age, requirement of neonatal oxygen supplementation, bronchopulmonary dysplasia and tracheal colonisation with Haemophilus influenzae are correlated with prolonged mechanical ventilation in children with bronchiolitis. Only bronchopulmonary dysplasia was associated with a need for extracorporeal membrane oxygenation that may provide lifesaving support in infants refractory to conventional management.