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In a situation with normal CBF and without increased energy utilization, increased glucose utilization (CMRglc) can be a sign of impaired mitochondrial metabolism, which may be an early step in the injury cascade during reperfusion after hypoxia–ischemia (HI). Seven-day-old rats underwent unilateral carotid artery ligation and 70 minutes of HI. At 3, 6, 12, 24, and 48 or 72 hours after the insult, the CMRglc was measured by the 2-deoxyglucose method, and CBF by the iodoantipyrine method. These were compared with hematoxylin-eosin staining and microtubule-associated protein 2 (MAP 2) immunostaining in adjacent sections. In the ipsilateral hemisphere, there appeared regions with increased CMRglc compared with the contralateral hemisphere 3 to 12 hours after HI that also showed partial loss of MAP 2 immunostaining and early ischemic changes. These areas receded, leaving central glucose hypoutilizing areas with complete loss of MAP 2 immunostaining and histologic infarction, surrounded by only a rim of tissue with increased CMRglc. At 24 and 72 hours after the insult, no regions with increased CMRglc remained. Despite loss of MAP 2 immunostaining and histologic signs of infarction at 24 hours, cortical CBF was not reduced until 48 hours after HI, whereas the CBF in the caudate-putamen already was decreased compared with the contralateral side at 3 hours after HI. In conclusion, early reperfusion is characterized by glucose hyperutilizing areas in the cerebral cortex, followed by a secondary phase with low CMRglc and infarction.

a Institute for Clinical Sciences, Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41685, Sweden b Centre for the Developing Brain, Department of Perinatal Imaging and Health, King's College London, St. Thomas' Hospital, London SE1 7EH, UK c Institute for Neuroscience and Physiology, Department of Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg 40530, Sweden

In adult rats N-methyl-D-aspartate receptor (NMDAR) antagonists increase glucose use and induce a 72-kD heat shock protein (HSP72) expression in limbic system areas that later undergo neuronal necrosis, which have limited the clinical development of these drugs. Dizocilpine maleate (MK-801) and magnesium sulfate (MgSO4) reduce hypoxic-ischemic brain injury in immature animals, but the effects on HSP72 expression and glucose use are unknown. Seven-day-old rats received injections of either vehicle (control), 0.5 or 1.0 mg/kg MK-801, or 2 or 4 mmol/kg MgSO4. Glucose utilization was measured with the deoxyglucose method, 30 min, 48 h, and 4 d after injection. HSP72 immunostaining was evaluated 4 or 24 h after injection. Both doses of MK-801 and 4 mmol/kg MgSO4 induced a temporary decrease in glucose use in the posterior cingulate and retrosplenial cortex, the CA1 and CA3 subfields of the hippocampus, the caudoputamen, and the parietal cortex. Doses of 2 mmol/kg MgSO4 did not affect glucose use in any structure. Neuronal HSP72 expression was not found in any drug-treated rats. In conclusion, neither MK-801 nor MgSO4 increased glucose use in the limbic system and did not induce HSP72 expression, suggesting that NMDAR antagonists lack direct neurotoxicity in the immature brain.

The Levene model in 7-day-old rats is the most often used model of hypoxia-ischaemia (HI) in immature animals. The rat central nervous system is immature at birth and corresponds neurodevelopmentally to the term human infant during the second postnatal week. The Levene model of HI differs from clinical asphyxia with respect to the unilateral distribution of brain injury and lack of multi-organ dysfunction. Furthermore, it does not allow cardiovascular monitoring or repeated blood sampling. On the other hand, the progressive nature of HI bears many similarities to birth asphyxia with regard to blood flow changes and cellular metabolic derangements. The model is well characterized, easy to carry out and the low cost allows inclusion of a sufficient number of animals for dose-response evaluation of neuroprotective agents. In addition, it provides the unique opportunity of long-term evaluation of neuropathological and functional outcome.

Intrauterine infection induces an intra-amniotic inflammatory response involving the activation of a number of cytokines and chemokines which, in turn, may trigger preterm contractions, cervical ripening and rupture of the membranes. Infection and cytokine-mediated inflammation appear to play a prominent role in preterm birth at early gestations (

Highlight ► A unique catalogue of phenotype markers and neuronotoxic effects of polarised primary microglia, as a comparative tool to screen neurotherapies., Microglia mediate multiple facets of neuroinflammation, including cytotoxicity, repair, regeneration, and immunosuppression due to their ability to acquire diverse activation states, or phenotypes. Modulation of microglial phenotype is an appealing neurotherapeutic strategy but a comprehensive study of classical and more novel microglial phenotypic markers in vitro is lacking. The aim of this study was to outline the temporal expression of a battery of phenotype markers from polarised microglia to generate an in vitro tool for screening the immunomodulatory potential of novel compounds. We characterised expression of thirty-one macrophage/microglial phenotype markers in primary microglia over time (4, 12, 36, and 72 h), using RT-qPCR or multiplex protein assay. Firstly, we selected Interleukin-4 (IL-4) and lipopolysaccharide (LPS) as the strongest M1–M2 polarising stimuli, from six stimuli tested. At each time point, markers useful to identify that microglia were M1 included iNOS, Cox-2 and IL-6 and a loss of M2a markers. Markers useful for quantifying M2b-immunomodulatory microglia included, increased IL-1RA and SOCS3 and for M2a-repair and regeneration, included increased arginase-1, and a loss of the M1 and M2b markers were discriminatory. Additional markers were regulated at fewer time points, but are still likely important to monitor when assessing the immunomodulatory potential of novel therapies. Further, to facilitate identification of how novel immunomodulatory treatments alter the functional affects of microglia, we characterised how the soluble products from polarised microglia affected the type and rate of neuronal death; M1/2b induced increasing and M2a-induced decreasing neuronal loss. We also assessed any effects of prior activation state, to provide a way to identify how a novel compound may alter phenotype depending on the stage of injury/insult progression. We identified generally that a prior M1/2b reduced the ability of microglia to switch to M2a. Altogether, we have characterised a profile of phenotype markers and a mechanism of assessing functional outcome that we can use as a reference guide for first-line screening of novel immunomodulatory therapies in vitro in the search for viable neuroprotectants.

The early postnatal period represents an important window in rodent hippocampal development with peak hilar neurogenesis and widespread microgliogenesis occurring in the first week of life. Inflammation occurring during this period may negatively influence development, potentially facilitating or increasing susceptibility to later-life pathology. We administered the Gram-negative bacterial coat protein lipopolysaccharide (LPS) systemically at postnatal day 5 (1 mg/kg i.p.) and assessed potential effects on microgliogenesis, inflammation and neurogenesis in the developing hippocampus. LPS administration led to an acute but transient increase in absolute number and density of ionized calcium-binding adaptor molecule 1-immunoreactive microglia, a change attributable to increased proliferation of central nervous system-resident microglia/microglial precursor cells but not infiltration of peripheral monocyte-derived macrophages. qRT-PCR analysis of hippocampal gene expression showed these LPS-mediated changes to be associated with persistent dysregulation of genes associated with both M1 and M2 microglial phenotypes, indicating prolonged alteration in hippocampal inflammatory status. Further, analysis of progenitor cell regulation in the hippocampal subgranular zone revealed a transient inhibition of the neuronal differentiation pathway up to 2 weeks after LPS administration, a change occurring specifically through effects on type 3 neural progenitor cells and independently of altered cell proliferation or survival of newly born cells. Together, our results show that systemic inflammation occurring during the early neonatal period is sufficient to alter inflammatory status and dysregulate the ongoing process of neurogenesis in the developing hippocampal germinal niche.

Background/Aims: Both excitotoxicity and neuroinflammation are associated with oxidative stress. One transcription factor, nuclear factor E2-related factor 2 (Nrf2), and one transcription cofactor, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), increase the endogenous antioxidant defence and can thus modulate neuronal cell death. Here, we investigated the temporal effects (after 24 and 72 h) of systemic (i.p.) administration of lipopolysaccharide (LPS) on the cerebral Nrf2 and PGC-1α systems. Methods and Results: Seven-day-old rat pups were injected with LPS (0.3 mg/kg). After 24 h, the protein levels of γ-glutamylcysteine ligase modulatory subunit, γ-glutamylcysteine ligase catalytic subunit, Nrf2, PGC-1α and manganese superoxide dismutase (MnSOD) were increased in parallel with decreased levels of Keap1. These effects were correlated with an increased level of phosphorylated Akt and elevated acetylation of histone 4. In contrast, 72 h following LPS, a decrease in the components of the Nrf2 system in parallel with an increase in Keap1 was observed. The down-regulation after 72 h correlated with phosphorylation of p38 mitogen-activated protein kinase, while there were no changes in PGC-1α and MnSOD protein levels or the acetylation/methylation pattern of histones. Conclusion: Systemic LPS in neonatal rats induced time-dependent changes in brain Nrf2 and PGC-1α that correlated well with the protective effect observed after 24 h (pre-conditioning) and the deleterious effects observed after 72 h (sensitizing) of systemic LPS reported earlier. Collectively, the results point towards Nrf2 and PGC-1α as a possible mechanism behind these effects. Fil: Correa, Fernando Gabriel. University of Gothenburg. Institute of Biomedicine; Suecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Ljunggren, Elin. University of Gothenburg. Institute of Biomedicine; Suecia Fil: Patil, Jaspal. University of Gothenburg. Institute of Biomedicine; Suecia Fil: Wang, Xiaoyang. University of Gothenburg. Institute of Neuroscience and Physiology; Suecia Fil: Hagberg, Henrik. University of Gothenburg. Institute of Neuroscience and Physiology; Suecia Fil: Mallard, Carina. University of Gothenburg. Institute of Neuroscience and Physiology; Suecia Fil: Sandberg, Mats. University of Gothenburg. Institute of Biomedicine; Suecia

BACKGROUND The periventricular white matter (PVWM) of the immature preterm brain is selectively vulnerable to a spectrum of injury. Although essential for normal brain development, the presence of resident microglia may exacerbate PVWM injury. METHODS We used immunohistochemistry to investigate microglia profile in human preterm noninjured control brains and in brains with evidence of germinal matrix hemorrhage/intraventricular hemorrhage (GMH/IVH), with median gestational age (GA) of 24.1 and 25.4 wk, respectively. RESULTS The number of microglia in the PVWM was higher than the other brain regions in both the control and GMH/IVH groups. Microglial density increased further in the PVWM of GMH/IVH brains, regardless of hemorrhage severity and despite normal macroscopic and imaging appearances to the PVWM. This was due to an increase in activated Iba1/CD68- and not Iba/CD45-immunopositive microglia. However, there were very few CD68/Ki67 colocalized cells, suggesting that the source of this increase may be due to a quick transformation of CD45-immunopositive hematopoietic microglia into CD68-immunopositive microglia. There was also increased apoptosis in the PVWM of all cases of GMH/IVH, with axonal injury and increased tumor necrosis factor-α (TNF-α) expression evident in the most severe cases. CONCLUSION Isolated GMH/IVH may influence ongoing brain development, with a significant role played by microglial activation.

There is currently no pharmacological treatment that provides protection against brain injury in neonates. It is known that activation of an innate immune response is a key, contributing factor in perinatal brain injury; therefore, the neuroprotective therapeutic potential of innate defense regulator peptides (IDRs) was investigated.The anti-inflammatory effects of 3 IDRs was measured in lipopolysaccharide (LPS)-activated murine microglia. IDRs were then assessed for their ability to confer neuroprotection in vivo when given 3 hours after neonatal brain injury in a clinically relevant model that combines an inflammatory challenge (LPS) with hypoxia-ischemia (HI). To gain insight into peptide-mediated effects on LPS-induced inflammation and neuroprotective mechanisms, global cerebral gene expression patterns were analyzed in pups that were treated with IDR-1018 either 4 hours before LPS or 3 hours after LPS+HI.IDR-1018 reduced inflammatory mediators produced by LPS-stimulated microglia cells in vitro and modulated LPS-induced neuroinflammation in vivo. When administered 3 hours after LPS+HI, IDR-1018 exerted effects on regulatory molecules of apoptotic (for, eg, Fadd and Tnfsf9) and inflammatory (for, eg, interleukin 1, tumor necrosis factor α, chemokines, and cell adhesion molecules) pathways and showed marked protection of both white and gray brain matter.IDR-1018 suppresses proinflammatory mediators and cell injurious mechanisms in the developing brain, and postinsult treatment is efficacious in reducing LPS-induced hypoxic-ischemic brain damage. IDR-1018 is effective in the brain when given systemically, confers neuroprotection of both gray and white matter, and lacks significant effects on the brain under normal conditions. Thus, this peptide provides the features of a promising neuroprotective agent in newborns with brain injury.

Post-treatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduces hypoxic–ischemic brain injury in immature animals. To elucidate possible mechanisms, cerebral glucose utilization (CMRglc) and cerebral blood flow (CBF) were measured 1–5 h after hypoxia–ischemia and administration of MK-801 in 7-day-old rats. After 100 min of unilateral hypoxia–ischemia, half of the pups were injected with MK-801. CMRglc was assessed by the [14C]deoxyglucose (2-DG) method. The brains were analyzed either by autoradiography or for energy metabolites and chromatographic separation of 2-DG-6-phosphate and 2-DG. CBF was measured by the autoradiographic [14C]iodoantipyrine method. Mean CMRglc in the cerebral cortex was increased ipsilaterally after hypoxia–ischemia to 15 ± 3.3 μmol 100 g−1 min−1 ( p < 0.01) and areas with CMRglc >20 μmol 100 g−1 min−1 amounted to 8.0 ± 7.7 mm2 in the ipsilateral hemisphere compared with 1.2 ± 1.6 mm2 contralateral ( p < 0.001). Treatment with MK-801 decreased CMRglc bilaterally ( p < 0.05) and reduced ipsilateral areas with increased CMRglc by 64% ( p < 0.01). CBF was unaltered after hypoxia–ischemia and by MK-801 treatment. In conclusion, regional glucose hyper-utilization in the parietal cortex after hypoxia–ischemia was attenuated by MK-801; this may have relevance to the neuroprotective effect of NMDA-receptor antagonists in this model.

Microglial and astrocyte responses to glucocorticoid pretreatment in the neonate exposed to hypoxia-ischemia (HI) are largely unknown. The expression of microglial antigens and astrocytic proliferation was compared in neonatal rats exposed to HI with and without cortisone. HI was induced in 7 day old rats. One group of rats received cortisone within 24 h of birth. Immunocytochemical and immunoblot investigations were performed. Monoclonal antibodies (OX18 and OX42) were used for the detection of the major histocompatibility complex (MHC) class I antigens and complement receptor 3 (CR3) respectively. Antibodies directed against glial fibrillary acidic protein (GFAP) and microtubule associated protein II (MAP II) were used to evaluate the extent of brain damage. Cortisone treatment provoked a decline in the number of microglial cells but did not modify GFAP levels in control rats which were not exposed to HI. Neuronal damage was similar in control and cortisone treated rats exposed to HI. There were also similarities in the expression of CR3 antigens on microglia. However microglial cells expressing MHC class I antigens were less prevalent in rats exposed to HI only. Cortisone pretreatment enhanced the expression of MHC class I antigens. Astrocytic proliferation was intense in rats exposed to HI; however in rats treated with cortisone and exposed to HI there was a drastic reduction in astrocytic proliferation. In conclusion it is suggested that microglia which survive cortisone pretreatment become over-activated thereby preventing astrocytic proliferation.

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery + 7.70% O2 for 100 min) and allowed to recover for 3 h, 24 h, 2 days or 14 days. Consecutive tissue sections were stained with antibodies against α-fodrin, the 150 kDa breakdown product of α-fodrin (FBDP, marker of calpain proteolysis) or microtubule associated protein 2 (MAP 2, marker of dendrosomatic neuronal injury). Cortical tissue pieces were subjected to Western blotting using the antibody against the FBDP. Areas with brain injury displayed a distinct loss of MAP 2 which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. A reciprocal staining pattern could be seen in the cerebral cortex, i.e. loss of MAP 2 and accumulation of FBDP, most pronounced 14 days after the insult. Fodrin and MAP 2 are known calpain substrates, and degradation of these proteins preceded neuronal degeneration, indicating that these proteases may be involved in the early events triggering the cascades leading to neuronal death.

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery +7.70% O 2 for 100 min). Ipsi-and contralateral parietal cerebral cortex was assayed with Western blotting for fodrin breakdown product (FBDP). Calpain immunoreactivity was assayed in the cytosolic fraction (CF) and the membrane and microsomal fraction (MMF). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62–68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This relative redistribution of calpains coincided with the appearance of FBDP in the left, ipsilateral hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204–292% of controls). No significant changes in FBDP could be detected in the right, contralateral hemisphere, indicating that although redistribution of calpains occurred, hypoxia per se did not suffice to initiate fodrin degradation in this model of neonatal hypoxic-ischemia.

Glial fibrillary acidic protein (GFAP) is the structural protein of intermediate filaments in astroglia. GFAP has extensively been used as a marker of gliosis in neuropathology. It also appears in excessive amounts in the cerebrospinal fluid in various acute brain disorders. Hypoxic-ischemic encephalopathy after perinatal asphyxia is a condition in which levels of GFAP could be expected to be elevated if brain cell damage occurs. We examined levels of GFAP by a sensitive ELISA in the cerebrospinal fluid of full-term infants between 12 and 48 h after birth. Cerebrospinal fluid-GFAP increased 5-fold in infants after perinatal asphyxia compared with a reference group (675 versus 137 ng/L, p < 0.001). The levels of GFAP also increased gradually in accordance with the severity of the neurologic symptoms ranked as degree of hypoxic-ischemic encephalopathy. We conclude that the cerebrospinal fluid levels of GFAP might be an important adjunct in the neonatal assessment of infants subject to perinatal asphyxia, and together with other neuronal or glial proteins, it might also help in defining temporal relationships in asphyxia.

The inflammatory response following hypoxic-ischemia (HI) in the neonate is largely unknown. Presently, the expression of microglial antigens and the beta-amyloid precursor protein (APP) were studied in relation to a dendrosomatic marker of neuronal injury (microtubule associated protein II; MAP II). HI was induced in 7-day-old rats by the combined unilateral carotid ligation and hypoxia. The pups (n = 23) were perfusion fixed 2-3 h, 24 h, 2-4 days and 14 days after HI and compared to sham-operated controls (n = 6). Antibodies were used for detection of the major histocompatibility complex II (OX-6), major histocompatibility complex I (OX-18) and complement receptor type 3 (OX-42), APP (APP 676-695) and MAP II (monoclonal MAP II) antigens. There was a transient APP expression 2-3 h after HI. A slight increase of microglial antigens (OX-18) was seen in the white matter 2 h after HI followed by a marked increase of OX-18, OX-6, OX-42 antigens 24 h-3-4 days in most injured regions with exception of the thalamus where a delayed (14 days) microglial response was seen. The latter event was parallelled by a delayed loss of MAP II. In conclusion, intense microglial expression occurs after neonatal HI either with an acute or delayed time-course depending on brain region.

Neuron specific enolase (NSE) in serum and cerebrospinal fluid (CSF) and glutamate in CSF were investigated in the immediate postasphyctic period in 22 term newborn infants. The cerebral function monitor (CFM) pattern was also assessed and hypoxic-ischaemic encephalopathy (HIE) was graded. NSE was significantly increased in the CSF of infants with HIE (median value 25.4 micrograms/l) compared with control infants (10.0 micrograms/l). Infants with the highest concentrations died. NSE in CSF correlated with the degree of asphyxial damage. Glutamate and NSE in CSF did not correlate, presumably due to the different time factors of the release after the insult. NSE in CSF corresponded well with the type of CFM pattern, which was also highly predictive of outcome.

To evaluate the maternal inflammatory response to microbial invasion of the amniotic cavity (MIAC) in women with preterm labor and preterm prelabor rupture of membranes using selected proteins in the maternal serum.A prospective cohort study.Labor ward from Salgrenska University Hospital. The evaluation of the maternal inflammatory response in the presence of MIAC in preterm labor and preterm prelabor rupture of membranes.One hundred and sixteen women with preterm labor and 73 women with preterm prelabor rupture of membranes between the gestational ages of 22(+0) and 33(+6) weeks.Twenty-seven maternal serum proteins were assayed by a multiple immunoassay.The maternal serum inflammatory response was evaluated according to the presence of MIAC. Data were stratified by gestational age.There were few differences in the maternal serum protein levels when MIAC was present in both preterm labor and preterm prelabor rupture of membranes. In preterm prelabor rupture of membranes, higher levels of interleukin-18 (median 654 vs. 361 pg/mL, p= 0.003) and lower levels of interleukin-1β (9.5 vs. 19.9 pg/mL, p= 0.008) and monocyte chemotactic protein-1 (139.1 vs. 212.6 pg/mL, p= 0.039) were observed in women with MIAC. Interleukin-6 (20.8 vs. 13.9 pg/mL, p= 0.019) was the only biomarker that increased significantly in preterm labor complicated with MIAC. All of the differences between preterm labor and preterm prelabor rupture of membranes were observed at less than 32(+0) weeks of gestation.A weak maternal inflammatory response in the serum was observed in women with MIAC.

International audience; INTODUCTION: Hypoxia-ischemia (HI) injury in term infants develops with a delay during the recovery phase, opening up a therapeutic window after the insult. Hypothermia is currently an established neuroprotective treatment in newborns with neonatal encephalopathy (NE), saving one in nine infants from developing neurological deficits. Caspase-2 is an initiator caspase, a key enzyme in the route to destruction and, therefore, theoretically a potential target for a pharmaceutical strategy to prevent HI brain damage. METHODS: The aim of this study was to explore the neuroprotective efficacy of hypothermia in combination with caspase-2 gene deficiency using the neonatal Rice-Vannucci model of HI injury in mice. RESULTS: HI brain injury was moderately reduced in caspase-2-imice as compared with wild-type (WT) mice. Five hours of hypothermia (33 degrees C) vs. normothermia (36 degrees C) directly after HI provided additive protection overall (temperature P = 0.0004, caspase-2 genotype P = 0.0029), in the hippocampus and thalamus, but not in other gray matter regions or white matter. Delayed hypothermia initiated 2 h after HI in combination with caspase-2 gene deficiency reduced injury in the hippocampus, but not in other brain areas. DISCUSSION: In conclusion, caspase-2 gene deficiency combined with hypothermia provided enhanced neuroprotection as compared with hypothermia alone.

To predict microbial invasion of the amniotic cavity (MIAC) and spontaneous preterm delivery within seven days using a panel of selected proteins from amniotic fluid in a Swedish population of preterm prelabor membrane rupture (PPROM).Prospective cohort study.Evaluation of intra-amniotic inflammation in preterm premature rupture of membranes.Sixty-six pregnant women with preterm prelabor membrane rupture at 22(+0) -33(+6) weeks' gestational age. Methods. Twenty-seven amniotic fluid proteins were assayed by a multiple immunoassay.The intra-amniotic inflammatory response was evaluated according to the presence of MIAC and the risk of spontaneous preterm delivery within seven days. A prediction model was constructed using logistic regression.The overall rates of MIAC and spontaneous preterm delivery within seven days were 20 and 50%, respectively. There was a higher inflammatory response in women with MIAC than in those without. Earlier gestational age at delivery and lower birthweight were observed in the presence of microbial invasion of the amniotic cavity. Amniotic fluid interleukin (IL)-6 and IL-10 were the best predictors of MIAC in terms of sensitivity (69%), specificity (81%), positive predictive value (47%), negative predictive value (91%) and a positive likelihood ratio of 3.6. There were no differences in intra-amniotic inflammatory response according to the risk of spontaneous preterm delivery within seven days.Amniotic fluid IL-6 and IL-10 are the best inflammatory biomarkers to predict MIAC in women with PPROM. Intra-amniotic inflammation does not predict the occurrence of spontaneous preterm delivery within seven days of PPROM.

We used quantitative in situ hybridization to study changes in the expression of c-fos following hypoxic-ischemia (H-I) in the neonatal rat brain. 7-day-old rat pups were subjected to a unilateral ligation of the common carotid artery followed by a 2 h 15 min hypoxic period (7.7% O2 in N2). This resulted in the expected ipsilateral infarction of cortex, lateral hippocampus, lateral-superior aspects of the striatum and the white matter of the corpus callosum. Brain damage was not seen in the contralateral hemisphere subjected only to hypoxia. c-fos mRNA levels increased in the contralateral hemisphere immediately after the hypoxia and had returned towards normal levels 2 h thereafter. In the ipsilateral hemisphere, the expression of c-fos was delayed but very marked at 2 h. Animals subjected only to hypoxia showed little or no increase in c-fos mRNA. Thus the earliest recorded increase in c-fos after hypoxic ischemia, which occurred on the non-ischemic, contralateral side, may represent a generalized response to a more localized insult.

This study addresses the issue of endpoint selection in the evaluation of neuroprotective drugs in experimental focal ischaemia. Previous work with the permanent middle cerebral artery (MCA) occlusion model in the rat has demonstrated that the ischaemic lesion does not acquire its final appearance until at least 28 days after the ictus. Therefore, the effect of the NMDA receptor blocker MK-801 (dizocilpine maleate) was evaluated both early (3 days) and late (28 days) after MCA occlusion to determine if the previously reported protective effect of a single post-ischaemic dose of MK-801 found in acute experiments remained after 28 days. Mk-801 (0.5 mg/kg, i.v.) or isotonic saline was randomly given to rats 30 min after MCA occlusion. Infarct volume and volume of ipsilateral and contralateral hemispheres were estimated from camera lucida drawings of 8 defined coronal histological sections of the brain. As expected, a 40% (p < 0.05) reduction of infarct size was found in MK-801 treated rats after 3 days. In animals evaluated 28 days after MCA occlusion, no significant difference in infarct size, total tissue loss (infarct volume+ipsilateral hemisphere atrophy) or remaining non-infarcted tissue (contralateral hemisphere--total tissue loss) was seen between the MK-801 and placebo treated rats. The results suggest that the single dose treatment with MK-801 postponed the evolution of the infarct, which at 3 days after MCA occlusion is still in progress, possibly by ameliorating oedema formation. It remains to be shown if a multiple dose treatment with NMDA receptor antagonists improves the final neuropathological outcome after experimental stroke.(ABSTRACT TRUNCATED AT 250 WORDS)

The extracellular calcium concentration ([Ca2+]ec) was recorded by calcium-sensitive microelectrodes in the parietal cortex of 9-11 day old rats during anoxia. During the first 10 min of anoxia, [Ca2+]ec increased from 1.1 mM to 1.5 +/- 0.23 mM, and thereafter it started to decrease reaching below basal level after around 13 min. The [Ca2+]ec decrease was either slow and continuous, or biphased with a rapid initial decrease followed by a continuous slow decrease. After 60 min of anoxia, the [Ca2+]ec had reached 0.2-0.3 mM. Changes in [Ca2+]ec in animals treated with the NMDA receptor antagonist MK-801 (0.3 mg/kg i.p.) did not display any significant differences compared to controls. Thus, the strong neuroprotective effect of MK-801 in ischemic situations in the immature brain can not be explained by a prevention of calcium entry during anoxic depolarization.

In a model of perinatal hypoxic-ischemic brain damage, we examined the neuroprotective efficacy of posttreatment with the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist NBQX. Unilateral brain damage developed in 95% of rat pups subjected to hypoxia-ischemia with a 27.8 ± 1.2% weight deficit of the damaged hemisphere. MK-801 in doses of 0.3 and 0.5 mg/kg i.p. reduced the brain damage by 61% (p < 0.001) and 43% (p < 0.001), respectively. A higher dose of MK-801 (0.75 mg/kg) did not offer neuroprotection. Treatment with NBQX (40 mg/kg) reduced the hemispheric lesion by 28% (p < 0.05). In conclusion, posttreatment with both NBQX and low doses of MK-801 reduced perinatal brain damage. The NMDA receptor antagonist offered stronger neuroprotection which is in agreement with a proposed NMDA receptor hyperactivity around postnatal day 7 in rats.

Mitochondria are key contributors to many forms of cell death including those resulting from neonatal hypoxic-ischemic brain injury. Mice have become increasingly popular in studies of brain injury, but there are few reports evaluating mitochondrial isolation procedures for the neonatal mouse brain. Using evaluation of respiratory activity, marker enzymes, western blotting and electron microscopy, we have compared a previously published procedure for isolating mitochondria from neonatal mouse brain (method A) with procedures adapted from those for adult rats (method B) and neonatal rats (method C). All three procedures use Percoll density gradient centrifugation as a key step in the isolation but differ in many aspects of the fractionation procedure and the solutions used during fractionation. Methods A and B both produced highly enriched fractions of well-coupled mitochondria with high rates of respiratory activity. The fraction from method C exhibited less preservation of respiratory properties and was more contaminated with other subcellular components. Method A offers the advantage of being more rapid and producing larger mitochondrial yields making it useful for routine applications. However, method B produced mitochondria that were less contaminated with synaptosomes and associated cytosolic components that suits studies that have a requirement for higher mitochondrial purification.

Inflammation is increasingly recognized as being of both physiological and pathological importance in the immature brain. The rationale of this review is to present an update on this topic with focus on long-term consequences of inflammation during childhood and in adults. The immature brain can be exposed to inflammation in connection with viral or bacterial infection during pregnancy or as a result of sterile central nervous system (CNS) insults. Through efficient anti-inflammatory and reparative processes, inflammation may resolve without any harmful effects on the brain. Alternatively, inflammation contributes to injury or enhances CNS vulnerability. Acute inflammation can also be shifted to a chronic inflammatory state and/or adversely affect brain development. Hypothetically, microglia are the main immunocompetent cells in the immature CNS, and depending on the stimulus, molecular context, and timing, these cells will acquire various phenotypes, which will be critical regarding the CNS consequences of inflammation. Inflammation has long-term consequences and could speculatively modify the risk of a variety of neurological disorders, including cerebral palsy, autism spectrum disorders, schizophrenia, multiple sclerosis, cognitive impairment, and Parkinson disease. So far, the picture is incomplete, and data mostly experimental. Further studies are required to strengthen the associations in humans and to determine whether novel therapeutic interventions during the perinatal period can influence the occurrence of neurological disease later in life.

Prenatal inflammation is considered an important factor contributing to preterm birth and neonatal mortality and morbidity. The impact of prenatal inflammation on fetal bioenergetic status and the correlation of specific metabolites to inflammatory-induced developmental brain injury are unknown. We used a global metabolomics approach to examine plasma metabolites differentially regulated by intrauterine inflammation. Preterm-equivalent sheep fetuses were randomized to i.v. bolus infusion of either saline-vehicle or LPS. Blood samples were collected at baseline 2 h, 6 h and daily up to 10 days for metabolite quantification. Animals were killed at 10 days after LPS injection, and brain injury was assessed by histopathology. We detected both acute and delayed effects of LPS on fetal metabolism, with a long-term down-regulation of fetal energy metabolism. Within the first 3 days after LPS, 121 metabolites were up-regulated or down-regulated. A transient phase (4-6 days), in which metabolite levels recovered to baseline, was followed by a second phase marked by an opposing down-regulation of energy metabolites, increased pO ₂ and increased markers of inflammation and ADMA. The characteristics of the metabolite response to LPS in these two phases, defined as 2 h to 2 days and at 6-9 days, respectively, were strongly correlated with white and grey matter volumes at 10 days recovery. Based on these results we propose a novel concept of inflammatory-induced hibernation of the fetus. Inflammatory priming of fetal metabolism correlated with measures of brain injury, suggesting potential for future biomarker research and the identification of therapeutic targets. © 2011 Keller et al.

Background and aims: Prenatal inflammation is currently being seen as one important factor of brain injury in preterm infants. Currently there is a lack of an early biomarker of brain injury. In addition it is not known how prenatal inflammation influences fetal metabolism in short and long-term. Here, we Methods: Fetuses were randomized to i.v. bolus infusion of either saline-vehicle (n=7) or 200ng E.coli LPS (n=9) at 102.5±0.5 days of gestation. Blood samples were collected at baseline 2h, 6h hours and daily up to 10 days for metabolite quantification. Metabolites were correlated to findings in MRI, EEG and histopathology. Results: LPS induced a two-phase pattern in metabolite changes. Within the first 3 days, 121 metabolites were impacted (immune response, energy metabolism and tissue injury). Thereafter, a transient period (4-6days) without changes compared to baseline, was followed by a second phase marked by an opposite regulation of energy metabolites and inflammatory markers. At the multivariate level, the two-phase characteristics of the metabolite response to LPS was strongly associated with white/grey matter volumes and the number of oligo-2 positive cells at 2h-to 2days and at 6-9days. Conclusions: In this first in-utero analysis we detected a significant impact of LPS on fetal metabolism in both the short and long-term. Thus we hypothesize that there is specific inflammatory priming of the fetal metabolism in the blood, as supported by the bi-phasic pattern of metabolites, which correlate with brain injury.

OBJECTIVE Preterm infants exhibit chronic deficits in white matter (WM) and cortical maturation. Although fetal infection/inflammation may contribute to WM pathology the factors contributing to cortical changes are largely unknown. We examined the effect of fetal lipopolysaccharide (LPS) exposure on WM and cortical development as assessed by magnetic resonance imaging (MRI) electroencephalography (EEG) and histopathology in fetal sheep at preterm human equivalent age. METHODS LPS was administered to fetal sheep at 102.5 ± 0.5 days of gestation. Continuous biophysical recordings were analyzed for 10 days after LPS. At postmortem measurement of cerebral WM and cortical tissue volumes was achieved by stereological techniques. Specific effects of LPS on MRI assessed T(1) weighted and T(2) weighted images and immunohistochemical expression of oligodendrocytes proliferating cells cortical NeuN positive and Nurr1 positive neurons (subplate marker) and cell death mechanisms were examined. RESULTS We observed reductions in WM (21; LPS 1.19 ± 0.04 vs control 1.51 ± 0.07 cm(3); p < 0.001) and cortical (18; LPS 2.34 ± 0.10 vs control 2.85 ± 0.07 cm(3); p < 0.001) volumes associated with overt and diffuse WM injury T(1) /T(2) weighted signal alterations and reduced numbers of WM oligodendrocytes (LPS 485 ± 31 vs control 699 ± 69 cells/mm(2); p = 0.0189) and NeuN positive (LPS 421 ± 71 vs control 718 ± 92 cells/mm(2); p = 0.04) and Nurr1 positive (control 2.5 ± 0.6 vs LPS 0.6 ± 0.1 cells/mm(2); p = 0.007) cortical neurons after LPS. Moreover there was loss of the normal maturational increase in cortical EEG amplitude which correlated with reduced cortical volumes. INTERPRETATION Fetal exposure to LPS prior to myelination onset can impair both white matter and cortical development in a preclinical large animal model supporting a role for maternal/fetal infection in the pathogenesis of preterm brain injury.

International audience; Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia–ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery plus 7.7% O2 for 2 h) and allowed to recover for 0 min, 30 min or 20 h. The calpain and calpastatin activities were assayed in subcellular fractions of the ipsilateral, hypoxic-ischemic and the contralateral, hypoxic hemisphere. An upregulation of calpain activity occurred in the hypoxic hemisphere, both in the major, cytosolic fraction and in the hypotonic, membrane associated fraction (110% and 133% of controls, respectively). The hypoxic-ischemic hemisphere displayed a decrease in calpain activity in the cytosolic fraction but an increase in the hypotonic fraction (90% and 111% of controls, respectively). The changes in calpastatin activity were less pronounced. This indicates that an upregulation of calpain activity occurs in parallel with development of hypoxic-ischemic damage. However, this upregulation is not necessarily coupled to development of injury as lesions are not seen in the hypoxic hemisphere.

Excitotoxic amino acids contain two acidic groups, but cysteine represents an exception to this rule. The hypothesis that cysteine toxicity is mediated by the oxidized and diacidic metabolites cysteine sulphinate and/or cysteate was tested in the present study. The issue was approached in three different ways. Firstly, the distribution of brain injury after subcutaneous administration of cysteine (1 mg/g) to 4-day-old rats was compared with that caused by cysteine sulphinate (3 mg/g). Secondly, the effects of excitatory amino acid receptor antagonists on cysteine and cysteine sulphinate toxicity were investigated. Thirdly, the cerebral concentrations of cysteine sulphinate were determined after cysteine administration and compared with those obtained after cysteine sulphinate injection. The cerebral cortex was the region most vulnerable to cysteine toxicity, followed by the hippocampus (especially the medial subicular neurons), amygdala, caudoputamen, cerebellum and septum. Pronounced extravasation of red blood cells was observed in lesioned areas. One day after cysteine administration, the injury was infarction-like and sharply demarcated. Cysteine sulphinate-induced damage resembled cysteine-induced lesions in some respects: the anterior cingulate and retrosplenial cortices, as well as medial subicular cells, were quite vulnerable. However, the differences prevailed. Cysteine sulphinate, but not cysteine, killed neurons of the superficial part of the tectum, the medial habenula, the ventromedial hypothalamus and the arcuate nucleus. Further, while cysteine toxicity was prominent in deep cortical layers, cysteine sulphinate preferentially damaged superficial cortical neurons. Cysteine toxicity was abolished by pretreatment with MK-801, a selective NMDA antagonist, but not by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, a selective AMPA receptor blocker. In contrast, the considerably smaller lesion seen after cysteine sulphinate administration was only partially prevented by MK-801. Large (19-fold) increases in cortical cysteine sulphinate concentration were noted after injection of a toxic dose of cysteine. This corresponds to 90 nmol cysteine sulphinate/g protein. The cysteate concentration was not increased above the detection limit. Injection of a toxic dose of cysteine sulphinate elevated cysteine sulphinate concentration in the frontomedial cortex (a region consistently injured by cysteine sulphinate) almost three orders of magnitude more than that observed after cysteine administration. Taken together, these results strongly suggest that neither cysteine sulphinate nor cysteate alone mediate cysteine toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Perinatal inflammation is a major risk factor for neurological deficits in preterm infants. Several experimental studies have shown that systemic inflammation can alter the programming of the developing brain. However, these studies do not offer detailed pathophysiological mechanisms, and they rely on relatively severe infectious or inflammatory stimuli that most likely do not reflect the levels of systemic inflammation observed in many human preterm infants. The goal of the present study was to test the hypothesis that moderate systemic inflammation is sufficient to alter white matter development.Newborn mice received twice-daily intraperitoneal injections of interleukin-1β (IL-1β) over 5 days and were studied for myelination, oligodendrogenesis, and behavior and with magnetic resonance imaging (MRI).Mice exposed to IL-1β had a long-lasting myelination defect that was characterized by an increased number of nonmyelinated axons. They also displayed a reduction of the diameter of the myelinated axons. In addition, IL-1β induced a significant reduction of the density of myelinating oligodendrocytes accompanied by an increased density of oligodendrocyte progenitors, suggesting a partial blockade in the oligodendrocyte maturation process. Accordingly, IL-1β disrupted the coordinated expression of several transcription factors known to control oligodendrocyte maturation. These cellular and molecular abnormalities were correlated with a reduced white matter fractional anisotropy on diffusion tensor imaging and with memory deficits.Moderate perinatal systemic inflammation alters the developmental program of the white matter. This insult induces a long-lasting myelination deficit accompanied by cognitive defects and MRI abnormalities, further supporting the clinical relevance of the present data.

Objective: Perinatal brain injury is a major cause of neurodevelopmental handicaps. Multiple pathways of oxidant stress, inflammation, and excitotoxicity lead to cell damage and death, including caspase-dependent apoptosis. Caspase-2 (Casp2; Nedd-2, Ich-1) is a developmentally regulated initiator caspase, which poorly cleaves other caspases but can initiate mitochondrial outer membrane permeabilization. We have investigated if Casp2 could mediate perinatal ischemic brain damage. Methods: Casp2 expression in human neonatal brains and developmental patterns in rats and mice were evaluated. Casp2-deficient (Casp2−/−), wild-type (WT), and heterozygous (Casp2+/−) newborn C57BL/6 mice were subjected to hypoxia-ischemia (unilateral carotid occlusion + exposure to 10% oxygen for 50 minutes) or intracerebral injection of the excitotoxic N-methyl-D-aspartate-receptor agonist ibotenate. In addition, Casp2 specific siRNAs were preinjected into the brain of WT newborn mice 24 hours before ibotenate treatment. Brain tissues were examined by immunohistochemical staining (cresyl violet, MAP2, NF68, Casp2, Casp3) and Western blotting. Lesion volumes and injury in the cortical plates and white matter were quantified together with activated Casp3. Results: Casp2 is highly expressed in the neonatal brain. Casp2-deficient mice subjected to hypoxia-ischemia at postnatal day 9 present significantly lower cerebral infarction, reduced white matter injury, and reduced Casp3 activation in the thalamus and hippocampus. Both Casp2−/− mice and siRNA-administered WT mice conferred reduction of gray and white matter injury after excitotoxic insult at postnatal day 5. Casp3 activation was also found reduced in Casp2-deficient mice subjected to excitotoxicity. Interpretation: These data suggest for the first time a role of Casp2 in neonatal brain damage. ANN NEUROL 2011

Perinatal hypoxic-ischemic encephalopathy can be a devastating complication of childbirth. Herein, the authors review the pathophysiology of hypoxic-ischemic encephalopathy and the current status of neuroprotective strategies to ameliorate the injury centering on four themes: (1) monitoring in the perinatal period, (2) rapid identification of affected neonates to allow timely institution of therapy, (3) preconditioning therapy (a therapeutic that reduces the brain vulnerability) before hypoxic-ischemic encephalopathy, and (4) prompt institution of postinsult therapies to ameliorate the evolving injury. Recent clinical trials have demonstrated the significant benefit for hypothermic therapy in the postnatal period; furthermore, there is accumulating preclinical evidence that adjunctive therapies can enhance hypothermic neuroprotection. Advances in the understanding of preconditioning may lead to the administration of neuroprotective agents earlier during childbirth. Although most of these neuroprotective strategies have not yet entered clinical practice, there is a significant hope that further developments will enhance hypothermic neuroprotection.

BACKGROUND Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP(-/-)Vim(-/-)) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP(-/-)Vim(-/-) mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown. METHODOLOGY/PRINCIPAL FINDINGS We subjected GFAP(-/-)Vim(-/-) and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP(-/-)Vim(-/-) and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN(+) neurons in the ischemic and contralateral hemisphere was comparable between GFAP(-/-)Vim(-/-) and wild-type mice. In wild-type mice, the number of S100(+) astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0+/-50.1 vs. 85.6+/-34.0, p

After 6–12 h of recovery from transient cerebral ischemia, the pyramidal cells of the hipppocampal CA1 region take up excessive amounts of calcium upon electrical stimulation, which has been suggested to be important for the development of delayed neuronal death. The aim of this study was to further characterize this enhanced calcium uptake with respect to time-course of development, relationship to neuronal damage, and amplitude of evoked field potentials as well as the dependency on N-methyl-d-aspartate (NMDA) and non-NMDA receptors. Adult Wistar rats were used and calcium-sensitive microelectrodes were placed in the stratum radiatum of the CA1 hippocampus for recording of the extracellular calcium concentration ([Ca2+]ec) during 20 min of ischemia and for 6 h of reflow. High-frequency stimulation of the perforant pathway elicited burst firing in CA1 and a transient decrease in [Ca2+]ec which reflects neuronal uptake. Shifts in [Ca2+]ec could not be evoked 0–1 h after ischemia. However, from 1–2 h burst firing could be evoked and the accompanying shift in [Ca2+]ec increased thereafter in amplitude with prolonged reflow, exceeded preischemic levels after 4 h, and reached 250 ± 116% (mean ± SD) of control after 6 h of reflow ( p < 0.05). The extracellular reference potential shift during electrical stimulation and the amplitude of evoked field potentials were still subnormal after 6 h [85 ± 25% and 83 ± 25%, respectively (mean ± SD)]. There was a significant correlation between the degree of stimulated calcium uptake at 6 h postischemia and the extent of CA1 damage evaluated 7 days after the ischemic insult ( r = 0.849; p < 0.001). The shifts in [Ca2+]ec were reduced by the NMDA antagonist MK-801 (0.5–2 mg/kg, i.v.) to approximately 50% of the initial level during both control and postischemic conditions ( p < 0.01). The non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[ F]quinoxaline (NBQX) (42 ± 13 mg/kg, i.p.; mean ± SD) decreased the amplitude of the evoked field potentials (to 30 ± 28% of control, p < 0.05) and completely abolished the evoked shifts in [Ca2+]ec. In conclusion, the uptake of calcium into CA1 pyramidal cells during electrical stimulation was enhanced already 4 h after ischemia in spite of the fact that other measures of excitability were subnormal. This calcium uptake correlated to the extent of CA1 pyramidal cell damage and was dependent on both NMDA and non-NMDA receptor activation.

Perinatal brain damage is a major clinical problem. Recent studies suggest that excitatory amino acids (EAAs) may be important for the development of hypoxic-ischemic brain injury in the newborn. Experimental work demonstrates that the immature brain is hypersensitive to the toxic effects EAA ('excitotoxicity'), hypoxic-ischemia is accompanied by an extracellular overflow of EAAs and hypoxic-ischemic brain damage is reduced by EAA receptor antagonists. Clinical investigations demonstrate the presence of EAA receptors in vulnerable areas of the newborn human brain and the concentrations of EAAs in the cerebrospinal fluid are higher in asphyxiated than in control infants. Clinical studies are warranted to evaluate the importance of excitotoxicity for development of brain lesions after severe asphyxia.

Preterm infants exposed to inflammation are at increased risk of white matter injury and/or cerebral palsy. To investigate the effect of chronic inflammation on the developing white matter, we administered low-dose lipopolysaccharide once a day from postnatal days 3 to 11, examined white matter changes at postnatal day 12, and monitored serum levels of insulin-like growth factor 1 and insulin-like factor binding protein-3. A single injection of lipopolysaccharide decreased the serum insulin-like growth factor 1 level but not the insulin-like factor binding protein-3 level. At postnatal day 12, quantification of immunohistochemical staining for axonal, myelin, and oligodendrocyte markers revealed impaired myelination in subcortical white matter. In addition, brain gray matter volume decreased and spleen and liver weight increased at postnatal day 12. These data suggest chronic subclinical inflammation hampers development of white and gray matter in early life, which may be associated with insulin-like growth factor 1 deficiency.

Apoptosis-related mechanisms are important in the pathophysiology of hypoxic–ischemic injury in the neonatal brain. Caspases are the major executioners of apoptosis, but there are a number of upstream players that influence the cell death pathways. The Bcl-2 family proteins are important modulators of mitochondrial permeability, working either to promote or prevent apoptosis. In this study we focused on the anti-apoptotic Bcl-2 protein after neonatal cerebral hypoxia–ischemia (HI) in 8-day-old rats. Bcl-2 translocated to nuclei and accumulated there over the first 24 h of reperfusion after HI, as judged by immunohistochemistry and immuno-electron microscopy. We also found that the total level of Bcl-2 decreased after HI in vivo and after ionophore challenge in cultured human neuroblastoma (IMR-32) cells in vitro. Furthermore, the Bcl-2 reduction was calpain-dependent, because it could be prevented by the calpain inhibitor CX295 both in vivo and in vitro, suggesting cross-talk between excitotoxic and apoptotic mechanisms.

Although the role of microglial activation in neural injury remains controversial, there is increasing evidence for a detrimental effect in the immature brain, which may occur in response to release of neurotoxic substances including pro-inflammatory cytokines. However, the signaling mechanisms involved in microglial-induced neuronal cell death are unclear. Microglia isolated from the brains of wild-type (WT) or MyD88 knockout (KO) mice were exposed to PBS or the TLR4-ligand LPS (100 ng/mL) for 2, 6, 14, or 24 h, and the microglia-conditioned medium (MCM) collected. Detection of multiple inflammatory molecules in MCM was performed using a mouse 22-plex cytokine microbead array kit. Primary neuronal cultures were supplemented with the 14 or 24 h MCM, and the degree of neuronal apoptosis examined after exposure for 24 h. Results showed a rapid and sustained elevation in multiple inflammatory mediators in the MCM of WT microglia exposed to LPS, which was largely inhibited in MyD88 KO microglia. There was a significant increase in apoptotic death measured at 24 h in cultured neurons exposed to CM from either 14 or 24 h LPS-stimulated WT microglia (p < .05 vs. WT control). By contrast, there was no increase in apoptotic death in cultured neurons exposed to CM from 14 or 24 h LPS-stimulated MyD88 KO microglia (p = .15 vs. MyD88 KO control). These data suggest that MyD88-dependent activation of microglia by LPS causes release of factors directly toxic to neurons.

Brain injury after hypoxic-ischemic encephalopathy often develops with delayed appearance, opening a therapeutic window. Clinical studies in newborns show that post-hypoxic-ischemic hypothermia improves outcome. This has generated renewed interest in the molecular mechanisms of hypoxic-ischemic brain injury. In this brief review, we propose that mitochondrial permeabilization is crucial for injury to advance beyond the point of no return. We suggest that excitatory amino acids, nitric oxide, inflammation, trophic factor withdrawal, and an increased pro- versus antiapoptotic Bcl-2 protein ratio will trigger Bax-dependent mitochondrial outer membrane permeabilization. Mitochondrial outer membrane permeabilization, in turn, elicits mitochondrial release of cytochrome C, apoptosis-inducing factor, second mitochondria-derived activator of caspase/Diablo, and HtrA2/Omi. Cytochrome C efflux activates caspase-9/-3, leading to DNA fragmentation. Apoptosis-inducing factor interacts with cyclophilin A and induces chromatinolysis. Blockage of mitochondrial outer membrane permeabilization holds promise as a strategy for perinatal brain protection.

Matrix metalloproteinase-12 (MMP-12) is expressed in the brain and is important for myelin formation in the developing brain, while MMP-12 deficiency protects against spinal cord injury in the adult, suggesting a role for MMP-12 after brain injury. However, the role of MMP-12 in neonatal hypoxic-ischemic brain injury is not known. The purpose of this study was to investigate the expression of MMP-12 in the brain after neonatal hypoxia-ischemia (HI). HI was induced by unilateral ligation of the left carotid artery followed by hypoxia (10% O2, 36°C) for 50 min in postnatal day 9 C57/Bl6J mice. At 24 and 72 h after HI, the mRNA and protein expression of MMP-12 were measured by real-time PCR and Western blot, respectively. Distribution and cellular expression of MMP-12 were examined by immunohistochemistry. At 72 h after HI, both MMP-12 mRNA and protein expression were significantly increased in the ipsilateral hemisphere compared to the contralateral hemisphere and sham-operated animals (p < 0.05). The extent of tissue loss in the ipsilateral hemisphere at 72 h after HI was positively correlated with the degree of MMP-12 protein expression (r = 0.900, p < 0.05). In nonischemic animals, immunohistochemical analysis demonstrated weak cytoplasmic MMP-12 immunoreactivity throughout the brain in cells which resembled neurons and stronger immunoreactivity was observed in blood vessels. After HI, the MMP-12 staining became more prominent in the ipsilateral hemisphere and immunohistochemical double-labeling experiments identified the MMP-12-positive cells as neurons, isolectin and Olig2-positive cells. This study demonstrates that MMP-12 is upregulated after HI, suggesting that MMP-12 may contribute to injury in the immature brain, similar to that seen in the adult.

To compare perinatal and maternal outcomes between elective induction of labor versus expectant management of pregnancies at 41 weeks and beyond.Systematic review and meta-analysis.We searched PubMed, CINAHL, Cochrane Database of Systematic Reviews (CDSR), Database of Abstracts of Reviews of Effectiveness (DARE) and PsycINFO (1980 to November, 2007). Inclusion criteria were systematic reviews and randomized controlled trials comparing elective induction of labor versus expectant management of pregnancies at 41 weeks and beyond. Three or more reviewers independently read and evaluated all selected studies. Data were extracted and analyzed using Review Manager Software.Perinatal mortality.Thirteen trials fulfilled the inclusion criteria for the meta-analysis. Elective induction of labor was not associated with lower risk of perinatal mortality compared to expectant management (relative risks (RR): 0.33; 95% confidence intervals (CI): 0.10-1.09). Elective induction was associated with a significantly lower rate of meconium aspiration syndrome (RR: 0.43; 95% CI: 0.23-0.79). More women randomized to expectant management were delivered by cesarean section (RR: 0.87; 95% CI: 0.80-0.96).The meta-analysis illustrated a problem with rare outcomes such as perinatal mortality. No individual study with adequate sample size has been published, nor would a meta-analysis based on the current literature be sufficient. The optimal management of pregnancies at 41 weeks and beyond is thus unknown.

Cyclophilin D (CypD), a regulator of the mitochondrial membrane permeability transition pore (PTP), enhances Ca2+-induced mitochondrial permeabilization and cell death in the brain. However, the role of CypD in hypoxic-ischemic (HI) brain injury at different developmental ages is unknown. At postnatal day (P) 9 or P60, littermates of CypD-deficient [knock-out (KO)], wild-type (WT), and heterozygous mice were subjected to HI, and brain injury was evaluated 7 d after HI. CypD deficiency resulted in a significant reduction of HI brain injury at P60 but worsened injury at P9. After HI, caspase-dependent and -independent cell death pathways were more induced in P9 CypD KO mice than in WT controls, and apoptotic activation was minimal at P60. The PTP had a considerably higher induction threshold and lower sensitivity to cyclosporin A in neonatal versus adult mice. On the contrary, Bax inhibition markedly reduced caspase activation and brain injury in immature mice but was ineffective in the adult brain. Our findings suggest that CypD/PTP is critical for the development of brain injury in the adult, whereas Bax-dependent mechanisms prevail in the immature brain. The role of CypD in HI shifts from a predominantly prosurvival protein in the immature to a cell death mediator in the adult brain.

Excitatory amino acids (EAAs) have been implicated to play a part in the development of hypoxic-ischemic brain injury in the neonate. The aim of the present study was to follow changes of intra- and extracellular (microdialysis) amino acids in the cerebral cortex in a model where cortical hypoxic-ischemic damage is produced consistently. Hypoxic-ischemia (unilateral ligation of the carotid artery + 2 h of exposure to 7.8% oxygen) caused a depletion of tissue ATP, phosphocreatine and glucose with a concomittant accumulation of AMP and lactic acid in cortical tissue. These changes were accompanied by a decrease of tissue aspartate and glutamine whereas the contents of gamma-aminobutyric acid (GABA), phenylalanine, leucine, isoleucine, valine and alanine increased. In the extracellular fluid GABA, glutamate, aspartate, taurine, glycine and alanine all increased multi-fold during hypoxic-ischemia. Aspartate and glutamate returned to near initial levels 2 h after the end of the insult, whereas the elevation of glycine persisted during recovery. In conclusion, the high extracellular levels of EAAs and glycine may exert injurious effects during and after hypoxic-ischemia.

This study addresses the possible involvement of an agonist-induced postischemic hyperactivity in the delayed neuronal death of the CA1 hippocampus in the rat. In two sets of experiments, dialytrodes were implanted into the CA1 either acutely or chronically (24 h of recovery). During 20 min of cerebral ischemia (four-vessel occlusion model) and 8 h of reflow, we followed extracellular amino acids and multiple-unit activity. Multiple-unit activity ceased within 20 sec of ischemia and remained zero during the ischemic insult and for the following 1 h of reflow. During ischemia, extracellular aspartate, glutamate, taurine, and gamma-aminobutyric acid increased in both acute and chronic experiments (seven- to 26-fold). Multiple-unit activity recovered to preischemic levels following 4-6 h of reflow. In the group with dialytrodes implanted acutely, the continuous increase in multiple-unit activity reached 110% of basal at 8 h of reflow. In the group with dialytrodes implanted chronically, multiple-unit activity recovered faster and reached 140% of control at 8 h, paralleled by an increase in extracellular aspartate (5.5-fold) and glutamate (twofold). In conclusion, the postischemic increase of excitatory amino acids and the recovery of the neuronal activity may stress the CA1 pyramidal cells, which could be detrimental in combination with, e.g., postsynaptic impairments.