Chronic changes in oligodendrocyte sub-populations after middle cerebral artery occlusion in neonatal mice.

Neonatal stroke is common and causes life-long motor and cognitive sequelae. Because neonates with stroke are not diagnosed until days-months after the injury, chronic targets for repair are needed. We evaluated oligodendrocyte maturity and myelination and assessed oligodendrocyte gene expression changes using single cell RNA sequencing (scRNA seq) at chronic timepoints in a mouse model of neonatal arterial ischemic stroke. Mice underwent 60 min of transient right middle cerebral artery occlusion (MCAO) on postnatal day 10 (p10) and received 5-ethynyl-2'-deoxyuridine (EdU) on post-MCAO days 3-7 to label dividing cells. Animals were sacrificed 14 and 28-30 days post-MCAO for immunohistochemistry and electron microscopy. Oligodendrocytes were isolated from striatum 14 days post-MCAO for scRNA seq and differential gene expression analysis. The density of Olig2+ EdU+ cells was significantly increased in ipsilateral striatum 14 days post-MCAO and the majority of oligodendrocytes were immature. Density of Olig2+ EdU+ cells declined significantly between 14 and 28 days post-MCAO without a concurrent increase in mature Olig2+ EdU+ cells. By 28 days post-MCAO there were significantly fewer myelinated axons in ipsilateral striatum. scRNA seq identified a cluster of "disease associated oligodendrocytes (DOLs)" specific to the ischemic striatum, with increased expression of MHC class I genes. Gene ontology analysis suggested decreased enrichment of pathways involved in myelin production in the reactive cluster. Oligodendrocytes proliferate 3-7 days post-MCAO and persist at 14 days, but fail to mature by 28 days. MCAO induces a subset of oligodendrocytes with reactive phenotype, which may be a therapeutic target to promote white matter repair.

Cerebral ischemia in the developing brain.

Brain ischemia affects all ages, from neonates to the elderly population, and is a leading cause of mortality and morbidity. Multiple preclinical rodent models involving different ages have been developed to investigate the effect of ischemia during different times of key brain maturation events. Traditional models of developmental brain ischemia have focused on rodents at postnatal day 7-10, though emerging models in juvenile rodents (postnatal days 17-25) indicate that there may be fundamental differences in neuronal injury and functional outcomes following focal or global cerebral ischemia at different developmental ages, as well as in adults. Here, we consider the timing of injury in terms of excitation/inhibition balance, oxidative stress, inflammatory responses, blood brain barrier integrity, and white matter injury. Finally, we review translational strategies to improve function after ischemic brain injury, including new ideas regarding neurorestoration, or neural repair strategies that restore plasticity, at delayed time points after ischemia.

Imaging of fetal ventriculomegaly.

Fetal ventriculomegaly is the most common central nervous system abnormality detected by prenatal imaging. It has a high association with other anomalies. Etiologies and prognoses for fetal ventriculomegaly range from normal outcomes to significant neurodevelopmental sequelae. In this paper, we review the development, terminology, pathogenesis, imaging and prognosis of fetal ventriculomegaly.

Experimental pediatric stroke shows age-specific recovery of cognition and role of hippocampal Nogo-A receptor signaling.

Ischemic stroke is a leading cause of death worldwide and clinical data suggest that children may recover from stroke better than adults; however, supporting experimental data are lacking. We used our novel mouse model of experimental juvenile ischemic stroke (MCAO) to characterize age-specific cognitive dysfunction following ischemia. Juvenile and adult mice subjected to 45-min MCAO, and extracellular field recordings of CA1 neurons were performed to assess hippocampal synaptic plasticity changes after MCAO, and contextual fear conditioning was performed to evaluate memory and biochemistry used to analyze Nogo-A expression. Juvenile mice showed impaired synaptic plasticity seven days after MCAO, followed by full recovery by 30 days. Memory behavior was consistent with synaptic impairments and recovery after juvenile MCAO. Nogo-A expression increased in ipsilateral hippocampus seven days after MCAO compared to contralateral and sham hippocampus. Further, inhibition of Nogo-A receptors reversed MCAO-induced synaptic impairment in slices obtained seven days after juvenile MCAO. Adult MCAO-induced impairment of LTP was not associated with increased Nogo-A. This study demonstrates that stroke causes functional impairment in the hippocampus and recovery of behavioral and synaptic function is more robust in the young brain. Nogo-A receptor activity may account for the impairments seen following juvenile ischemic injury.

Subpial Hemorrhage of the Neonate.

Background and Purpose- Subpial hemorrhage of the neonate is a rare stroke subtype reported in few case series. Birth trauma and coagulopathy are commonly proposed etiologies. We evaluated our subpial hemorrhage of the neonate patient cohort to expand current understanding Methods- Cases of subpial hemorrhage of the neonate were identified by keyword searches of the institutional database. The medical records and magnetic resonance imagings were reviewed. Results- Seventeen cases were identified. Assisted delivery occurred in 12% of cases, and acute coagulation abnormalities occurred in 77%. Subpial hemorrhage of the neonate was located in the temporal lobe in 82%, with cytotoxic edema and medullary vein congestion and thrombosis subjacent to the hemorrhages in 100% and 76% of cases, respectively. Neurological disability was present in 44% of survivors. Three patients had chronic coagulation abnormalities. Conclusions- In our cohort, clinical findings supporting a potential relationship with birth trauma were infrequent. The imaging findings suggest a nonarterial, deep venous pattern of hemorrhagic ischemia.

Oligodendrocyte Progenitor Cell Proliferation and Fate after White Matter Stroke in Juvenile and Adult Mice.

The incidence of stroke in children is 2.4 per 100,000 person-years and results in long-term motor and cognitive disability. In ischemic stroke, white matter (WM) is frequently injured, but is relatively understudied compared to grey matter injury. Previous research suggests that the cellular response to WM ischemic injury is different at different ages. Little is known about whether WM repair mechanisms differ in children and adults. We utilized a model of focal ischemic WM injury to determine the oligodendrocyte (OL) response to focal WM ischemic injury in juvenile and adult mice. Methods: Juvenile (21-25 days of age) versus adult (2-3 months of age) mice underwent stereotaxic injection of the potent vasoconstrictor N5-(1-iminoethyhl)-L-ornithine (L-NIO) into the lateral corpus callosum (CC). Animals were sacrificed on postoperative day 3 (acute) or 21 (chronic). Cell birth-dating was performed acutely after WM stroke with 5-ethynyl-2-deoxyuridine (EdU) injected intraperitoneally. Immunohistochemistry was performed, as well as stereology, to measure injury volume. The acute oligodendrocyte progenitor cell (OPC) proliferation and the chronic OL cell fate were determined with immunohistochemistry. Compound action potentials were measured in the CC at acute and chronic time points. Results: Acutely WM injury volume was smaller in juveniles. There was significantly greater OPC proliferation in juvenile animals (acute) compared to adults, but newly born OLs did not survive and mature into myelinating cells at chronic time points. In addition, juveniles did not have improved histological or functional recovery when compared to adults. Protecting newly born OPCs is a potential therapeutic target in children with ischemic stroke.

Delayed inhibition of tonic inhibition enhances functional recovery following experimental ischemic stroke.

The current study focuses on the ability to improve cognitive function after stroke with interventions administered at delayed/chronic time points. In light of recent studies demonstrating delayed GABA antagonists improve motor function, we utilized electrophysiology, biochemistry and neurobehavioral methods to investigate the role of α5 GABAA receptors on hippocampal plasticity and functional recovery following ischemic stroke. Male C57Bl/6 mice were exposed to 45 min transient middle cerebral artery occlusion and analysis of synaptic and functional deficits performed 7 or 30 days after recovery. Our findings indicate that hippocampal long-term potentiation (LTP) is impaired 7 days after stroke and remain impaired for at least 30 days. We demonstrate that ex vivo administration of L655,708 reversed ischemia-induced plasticity deficits and importantly, in vivo administration at delayed time-points reversed stroke-induced memory deficits. Western blot analysis of hippocampal tissue reveals proteins responsible for GABA synthesis are upregulated (GAD65/67 and MAOB), increasing GABA in hippocampal interneurons 30 days after stroke. Thus, our data indicate that both synaptic plasticity and memory impairments observed after stroke are caused by excessive tonic GABA activity, making inhibition of specific GABA activity at delayed timepoints a potential therapeutic approach to improve functional recovery and reverse cognitive impairments after stroke.

Seizure Severity Is Correlated With Severity of Hypoxic-Ischemic Injury in Abusive Head Trauma.

BACKGROUND: The objective of this study was to characterize hypoxic-ischemic injury and seizures in abusive head trauma. METHODS: We studied 58 children with moderate or severe traumatic brain injury due to abusive head trauma. Continuous electroencephalograms and magnetic resonance images were scored. RESULTS: Electrographic seizures (51.2%) and hypoxic-ischemic injury (77.4%) were common in our cohort. Younger age was associated with electrographic seizures (no seizures: median age 13.5 months, interquartile range five to 25 months, versus seizures: 4.5 months, interquartile range 3 to 9.5 months; P = 0.001). Severity of hypoxic-ischemic injury was also associated with seizures (no seizures: median injury score 1.0, interquartile range 0 to 3, versus seizures: 4.5, interquartile range 3 to 8; P = 0.01), but traumatic injury severity was not associated with seizures (no seizures: mean injury score 3.78 ± 1.68 versus seizures: mean injury score 3.83 ± 0.95, P = 0.89). There was a correlation between hypoxic-ischemic injury severity and seizure burden when controlling for patient age (rs=0.61, P < 0.001). The ratio of restricted diffusion volume to total brain volume (restricted diffusion ratio) was smaller on magnetic resonance imaging done early (median restricted diffusion ratio 0.03, interquartile range 0 to 0.23 on magnetic resonance imaging done within two days versus median restricted diffusion ratio 0.13, interquartile range 0.01 to 0.43 on magnetic resonance imaging done after two days, P = 0.03). CONCLUSIONS: Electrographic seizures are common in children with moderate to severe traumatic brain injury from abusive head trauma, and therefore children with suspected abusive head trauma should be monitored with continuous electroencephalogram. Severity of hypoxic-ischemic brain injury is correlated with severity of seizures, and evidence of hypoxic-ischemic injury on magnetic resonance imaging may evolve over time. Therefore children with a high seizure burden should be reimaged to evaluate for evolving hypoxic-ischemic injury.

Etiology and treatment of arterial ischemic stroke in children and young adults.

OPINION STATEMENT: Stroke is the second leading cause of death worldwide (Go et al. Circulation 129:e28-292, 2014) and is a major cause of morbidity and mortality. Compared with older adults, arterial ischemic stroke (AIS) is relatively uncommon in children and young adults, comprising 5-10 % of all stroke (Biller Nat Rev Cardiol 6:395-97, 2009), but is associated with significant cost. In contrast to the declining overall incidence of stroke, some early studies suggest that the rate of stroke hospitalizations in children and young adults is rising (George et al. Ann Neurol 70:713-21, 2011; Kissela et al. Stroke 41:e224, 2010; Nguyen-Huynh et al. Stroke 43, 2012), emphasizing the importance of understanding the similarities and differences in etiology and treatment of AIS across the age spectrum. Among the most common causes of AIS in children are cardioembolism (often related to congenital heart disease), cervicocephalic arterial dissections, focal arteriopathy of childhood and several genetic and metabolic disorders, such as sickle cell disease (SCD). AIS in young adults is less well understood, but likely overlaps in etiology with both children and older adults. Young adults with AIS often have classic atherosclerotic risk factors similar to older adults, but are also more likely to have thrombophilias, cervicocephalic arterial dissections and cardioembolism, similar to children with AIS. Since little evidence exists regarding both acute treatment and secondary prevention after AIS in children and young adults, standard treatment practices are mainly extrapolated from research done in older adults. In most cases we recommend treating young adults per the guidelines published by the American Heart Association for adults with stroke (Jauch et al. Stroke 44:870-947, 2013; Kernan et al. Stroke 45:2160-2236, 2014) and children per the equivalent guidelines regarding pediatric stroke (Roach et al. Stroke 39:2644-91, 2008). It is also important in children and young adults to consider less common structural, metabolic and genetic risk factors for stroke, which may require more specific treatment. Other standard risk factors for stroke, including hypertension, hyperlipidemia and diabetes mellitus should also be addressed, but are less likely in children and young adults. Given the lack of data and possibility of rare underlying etiologies such as Antiphospholipid Antibody Syndrome or Ehlers-Danlos syndrome, we recommend including multiple specialists in the care of these patients, such as hematologists and vascular neurologists.

Placental TNF-α signaling in illness-induced complications of pregnancy.

Maternal infections are implicated in a variety of complications during pregnancy, including pregnancy loss, prematurity, and increased risk of neurodevelopmental disorders in the child. Here, we show in mice that even mild innate immune activation by low-dose lipopolysaccharide in early pregnancy causes hemorrhages in the placenta and increases the risk of pregnancy loss. Surviving fetuses exhibit hypoxia in the brain and impaired fetal neurogenesis. Maternal Toll-like receptor 4 signaling is a critical mediator of this process, and its activation is accompanied by elevated proinflammatory cytokines in the placenta. We evaluated the role of tumor necrosis factor-α (TNF-α) signaling and show that TNF receptor 1 (TNFR1) is necessary for the illness-induced placental pathology, accompanying fetal hypoxia, and neuroproliferative defects in the fetal brain. We also show that placental TNFR1 in the absence of maternal TNFR1 is sufficient for placental pathology to develop and that a clinically relevant TNF-α antagonist prevents placental pathology and fetal loss. Our observations suggest that the placenta is highly sensitive to proinflammatory signaling in early pregnancy and that TNF-α is an effective target for preventing illness-related placental defects and related risks to the fetus and fetal brain development.

Macrophages are comprised of resident brain microglia not infiltrating peripheral monocytes acutely after neonatal stroke.

Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia-reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post-natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h post-reperfusion to distinguish invading monocytes (CD45high/CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1-24 h post-reperfusion using ELISA-based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3-fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro-inflammatory cytokine IL-beta and chemokines cytokine-induced neutrophil chemoattractant protein 1 (CINC-1) and monocyte-chemoattractant protein 1 (MCP-1), first in systemic circulation and then in injured brain. Double immunofluorescence with cell-type specific markers showed that multiple cell types in the injured brain produce MCP-1. Our findings show that despite profound increases in MCP-1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia.

Aminoguanidine inhibits caspase-3 and calpain activation without affecting microglial activation following neonatal transient cerebral ischemia.

Microglial cells, the resident macrophages of the CNS, can be both beneficial and detrimental to the brain. These cells play a central role as mediators of neuroinflammation associated with many neurodegenerative states, including cerebral ischemia. Because microglial cells are both a major source of inducible nitric oxide synthase (iNOS)/nitric oxide (NO) production locally in the injured brain and are activated by NO-mediated injury, we tested whether iNOS inhibition reduces microglial activation and ischemic injury in a neonatal focal ischemia-reperfusion model. Post-natal day 7 rats were subjected to a 2 h transient middle cerebral artery (MCA) occlusion. Pups with confirmed injury on diffusion-weighted magnetic resonance imaging (MRI) during occlusion were administered 300 mg/kg/dose aminoguanidine (AG) or vehicle at 0, 4 and 18 h after reperfusion, and animals were killed at 24 or 72 h post-reperfusion. The effect of AG on microglial activation as judged by the acquisition of ED1 immunoreactivity and proliferation of ED1-positive cells, on activation of cell death pathways and on injury volume, was determined. The study shows that while AG attenuates caspase 3 and calpain activation in the injured tissue, treatment does not affect the rapidly occurring activation and proliferation of microglia following transient MCA occlusion in the immature rat, or reduce injury size.

Magnetic resonance imaging as a surrogate measure for histological sub-chronic endpoint in a neonatal rat stroke model.

INTRODUCTION: It is becoming increasingly recognized that CNS immaturity at birth affects ischemic injury and recovery, and that the consequences of neonatal stroke need to be studied using age-appropriate focal stroke models. The inclusion of magnetic resonance imaging (MRI) as a surrogate measure of stroke progression has provided useful information in adult models, but the benefit for neonatal stroke studies is yet to be established. METHODS: Postnatal 7-day (P7) rats were subjected to a 3-h transient occlusion of the middle cerebral artery (MCA) which was produced either by inserting a filament via the external carotid artery or via the internal carotid artery. MRI was used to delineate the size and pattern of injury acutely, during MCA occlusion, and 7 days following reperfusion. RESULTS: The size of the diffusion-weighted (DW) MRI-detectable injury during MCA occlusion was similar following both surgical procedures and resulted in histological lesions 7 days later in all animals. The extent of spontaneous recovery in individual animals varied substantially 7 days later within each group, as was depicted by a combination of DW- and T2W-MRI and confirmed by the corresponding histology. CONCLUSIONS: The ability of MRI to provide accurate information on the size of histological outcome at 7 days after neonatal focal transient ischemia suggests that MRI is useful as an intermediate surrogate measure of injury progression in long-term neonatal stroke studies.

Minocycline confers early but transient protection in the immature brain following focal cerebral ischemia-reperfusion.

The incidence of neonatal stroke is high and currently there are no strategies to protect the neonatal brain from stroke or reduce the sequelae. Agents capable of modifying inflammatory processes hold promise. We set out to determine whether delayed administration of one such agent, minocycline, protects the immature brain in a model of transient middle cerebral artery (MCA) occlusion in 7-day-old rat pups. Injury volume in minocycline (45 mg/kg/dose, beginning at 2 h after MCA occlusion) and vehicle-treated pups was determined 24 h and 7 days after onset of reperfusion. Accumulation of activated microglia/macrophages, phosphorylation of mitogen-activated protein kinase (MAPK) p38 in the brain, and concentrations of inflammatory mediators in plasma and brain were determined at 24 h. Minocycline significantly reduced the volume of injury at 24 h but not 7 days after transient MCA occlusion. The beneficial effect of minocycline acutely after reperfusion was not associated with changed ED1 phenotype, nor was the pattern of MAPK p38 phosphorylation altered. Minocycline reduced accumulation of IL-1beta and CINC-1 in the systemic circulation but failed to affect the increased levels of IL-1beta, IL-18, MCP-1 or CINC-1 in the injured brain tissue. Therefore, minocycline provides early but transient protection, which is largely independent of microglial activation or activation of the MAPK p38 pathway.