Pelin Cengiz

Central nervous system complications during pediatric extracorporeal life support: incidence and risk factors.

Objective:Identify the incidence and risk factors for development of acute, severe central nervous system (CNS) complications of pediatric extracorporeal life support (ECLS). Design:Retrospective review of Extracorporeal Life Support Organization (ELSO) registry database. Setting:Pediatric intensive care units of 115 tertiary centers internationally. Patients:Pediatric patients, 1 month to 18 yrs of age, who had ECLS between the years 1981–2002. Measurements and Main Results:Data concerning 4,942 patients who underwent one run of ECLS were analyzed. Six hundred thirty-six patients (12.9%) developed acute, severe CNS complications. Patients who required ECLS during extracorporeal cardiopulmonary resuscitation (n = 161; 3.3%) were more likely to develop CNS complications (n = 42; 26.1%) than patients who did not have extracorporeal cardiopulmonary resuscitation (p < .001; odds ratio [OR], 2.48; 95% confidence interval [CI], 1.73–3.57). Stepwise logistic regression analysis of therapies patients received before initiation of ECLS showed that the use of a left ventricular assist device (p = .001; OR, 3.45; 95% CI, 1.64–7.22), bicarbonate (p < .001; OR, 1.61; 95% CI, 1.26–2.05), and vasopressor/inotropic medications (p = .035; OR, 1.22; 95% CI, 1.01–1.48) were significant independent predictors of development of CNS complications. Among patients who had pulmonary failure as an indication for ECLS, the CNS complication rate was significantly higher for those treated with venoarterial ECLS than those who had venovenous ECLS (13.5% vs. 5.7%; p < .001; OR, 0.43; 95% CI, 0.34–0.67). Multiple logistic regression analysis of the complications other than CNS complications associated with the use of ECLS showed that pH <7.20, creatinine concentration >3.0 mg/dL, use of inotropes, presence of myocardial stun, and requirement of cardiopulmonary resuscitation during ECLS independently predicted development of CNS complications. Conclusion:Patients who have metabolic acidosis, a bicarbonate or inotrope/vasopressor requirement, cardiopulmonary resuscitation, or a left ventricular assist device before initiation of ECLS are at greater risk for development of CNS complications. After initiation of ECLS, patients who develop renal failure or metabolic acidosis or undergo venoarterial ECLS should be closely monitored for development of CNS complications.

Inhibition of Na+/H+ exchanger isoform 1 is neuroprotective in neonatal hypoxic ischemic brain injury.

We investigated the role of Na(+)/H(+) exchanger isoform 1 (NHE-1) in neonatal hypoxia/ischemia (HI). HI was induced by unilateral ligation of the left common carotid artery in postnatal day 9 (P9) mice, and subsequent exposure of animals to 8% O(2) for 55 min. A pre/posttreatment group received a selective and potent NHE-1 inhibitor HOE 642 (0.5 mg/kg, intraperitoneally) 5 min before HI, then at 24 and 48 h after HI. A posttreatment group received HOE 642 (0.5 mg/kg) at 10 min, 24 h, and 48 h after HI. Saline injections were used as vehicle controls. The vehicle-control brains at 72 h after HI exhibited neuronal degeneration in the ipsilateral hippocampus, striatum, and thalamus, as identified with Fluoro-Jade C positive staining and loss of microtubule-associated protein 2 (MAP2) expression. NHE-1 protein was upregulated in glial fibrillary acidic protein-positive reactive astrocytes. In HOE 642-treated brains, the morphologic hippocampal structures were better preserved and displayed less neurodegeneration and a higher level of MAP2 expression. Motor-learning deficit was detected at 4 weeks of age after HI in the vehicle control group. Inhibition of NHE-1 in P9 mice not only reduced neurodegeneration during the acute stage of HI but also improved the striatum-dependent motor learning and spatial learning at 8 weeks of age after HI. These findings suggest that NHE-1-mediated disruption of ionic homeostasis contributes to striatal and CA1 pyramidal neuronal injury after neonatal HI.

Chronic Neurological Deficits in Mice after Perinatal Hypoxia and Ischemia Correlate with Hemispheric Tissue Loss and White Matter Injury Detected by MRI

We investigated the effects of perinatal hypoxia-ischemia (HI) on brain injury and neurological functional outcome at postnatal day (P)30 through P90. HI was induced by exposing P9 mice to 8% O2 for 55 min using the Vannucci HI model. Following HI, mice were treated with either vehicle control or Na+/H+ exchanger isoform 1 (NHE1) inhibitor HOE 642. The animals were examined by the accelerating rotarod test at P30 and the Morris water maze (MWM) test at P60. T2-weighted MRI was conducted at P90. Diffusion tensor imaging (DTI) was subsequently performed in ex vivo brains, followed by immunohistochemical staining for changes in myelin basic protein (MBP) and neurofilament protein expression in the corpus callosum (CC). Animals at P30 after HI showed deficits in motor and spatial learning. T2 MRI detected a wide spectrum of brain injury in these animals. A positive linear correlation was observed between learning deficits and the degree of tissue loss in the ipsilateral hemisphere and hippocampus. Additionally, CC DTI fractional anisotropy (FA) values correlated with MBP expression. Both FA and MBP values correlated with performance on the MWM test. HOE 642-treated mice exhibited improved spatial learning and memory, and less white matter injury in the CC. These findings suggest that HI-induced cerebral atrophy and CC injury contribute to the development of deficits in learning and memory, and that inhibition of NHE1 is neuroprotective in part by reducing white matter injury. T2-weighted MRI and DTI are useful indicators of functional outcome after perinatal HI.

TrkB receptor agonist 7, 8 dihydroxyflavone triggers profound gender- dependent neuroprotection in mice after perinatal hypoxia and ischemia.

In this study, we investigated the effects of a bioactive high-affinity TrkB receptor agonist 7,8- dihydroxyflavone (7,8 DHF) on neonatal brain injury in female and male mice after hypoxia ischemia (HI). HI was induced by exposure of postnatal day 9 (P9) mice to 10% O2 for 50 minutes at 37°C after unilateral ligation of the left common carotid artery. Animals were randomly assigned to HI-vehicle control group [phosphate buffered saline (PBS), intraperitoneally (i.p.)] or HI + 7,8 DHF-treated groups (5 mg/kg in PBS, i.p at 10 min, 24 h, or with subsequent daily injections up to 7 days after HI). The HI-vehicle control mice exhibited neuronal degeneration in the ipsilateral hippocampus and cortex with increased Fluoro-Jade C positive staining and loss of microtubule associated protein 2 expression. In contrast, the 7,8 DHF-treated mice showed less hippocampal neurodegeneration and astrogliosis, with more profound effects in female than in male mice. Moreover, 7,8 DHF-treated mice improved motor learning and spatial learning at P30-60 compared to the HI-vehicle control mice. Diffusion tensor imaging of ex vivo brain tissues at P90 after HI revealed less reduction of fractional anisotropy values in the ipsilateral corpus callosum of 7,8 DHF-treated brains, which was accompanied with better preserved myelin basic protein expression and CA1 hippocampal structure. Taken together, these findings strongly suggest that TrkB agonist 7,8 DHF is protective against HI-mediated hippocampal neuronal death, white matter injury, and improves neurological function, with a more profound response in female than in male mice.

Stimulation of Na + /H + Exchanger Isoform 1 Promotes Microglial Migration

Regulation of microglial migration is not well understood. In this study, we proposed that Na+/H+ exchanger isoform 1 (NHE-1) is important in microglial migration. NHE-1 protein was co-localized with cytoskeletal protein ezrin in lamellipodia of microglia and maintained its more alkaline intracellular pH (pHi). Chemoattractant bradykinin (BK) stimulated microglial migration by increasing lamellipodial area and protrusion rate, but reducing lamellipodial persistence time. Interestingly, blocking NHE-1 activity with its potent inhibitor HOE 642 not only acidified microglia, abolished the BK-triggered dynamic changes of lamellipodia, but also reduced microglial motility and microchemotaxis in response to BK. In addition, NHE-1 activation resulted in intracellular Na+ loading as well as intracellular Ca2+ elevation mediated by stimulating reverse mode operation of Na+/Ca2+ exchange (NCXrev). Taken together, our study shows that NHE-1 protein is abundantly expressed in microglial lamellipodia and maintains alkaline pHi in response to BK stimulation. In addition, NHE-1 and NCXrev play a concerted role in BK-induced microglial migration via Na+ and Ca2+ signaling.

Endoplasmic reticulum Ca2+ signaling and mitochondrial Cyt c release in astrocytes following oxygen and glucose deprivation.

J. Neurochem. (2010) 114, 1436–1446.

Sustained Na+/H+ exchanger activation promotes gliotransmitter release from reactive hippocampal astrocytes following oxygen-glucose deprivation.

Hypoxia ischemia (HI)-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na+/H+ exchanger isoform 1 (NHE1) protein in mouse reactive hippocampal astrocyte function in an in vitro ischemia model (oxygen/glucose deprivation and reoxygenation, OGD/REOX). 2 h OGD significantly increased NHE1 protein expression and NHE1-mediated H+ efflux in hippocampal astrocytes. NHE1 activity remained stimulated during 1–5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na+ and Ca2+ overload. The latter was mediated by reversal of Na+/Ca2+ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα) during 1–24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na+ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H+ homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na+ and Ca2+ homeostasis, which reduces Na+-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI.

ERα Signaling Is Required for TrkB-Mediated Hippocampal Neuroprotection in Female Neonatal Mice after Hypoxic Ischemic Encephalopathy(1,2,3).

Abstract Male neonate brains are more susceptible to the effects of perinatal asphyxia resulting in hypoxia and ischemia (HI)-related brain injury. The relative resistance of female neonatal brains to adverse consequences of HI suggests that there are sex-specific mechanisms that afford females greater neuroprotection and/or facilitates recovery post-HI. We hypothesized that HI preferentially induces estrogen receptor α (ERα) expression in female neonatal hippocampi and that ERα is coupled to Src family kinase (SFK) activation that in turn augments phosphorylation of the TrkB and thereby results in decreased apoptosis. After inducing the Vannucci’s HI model on P9 (C57BL/6J) mice, female and male ERα wild-type (ERα+/+) or ERα null mutant (ERα−/−) mice received vehicle control or the selective TrkB agonist 7,8-dihydroxyflavone (7,8-DHF). Hippocampi were collected for analysis of mRNA of ERα and BDNF, protein levels of ERα, p-TrkB, p-src, and cleaved caspase 3 (c-caspase-3) post-HI. Our results demonstrate that: (1) HI differentially induces ERα expression in the hippocampus of the female versus male neonate, (2) src and TrkB phosphorylation post-HI is greater in females than in males after 7,8-DHF therapy, (3) src and TrkB phosphorylation post-HI depend on the presence of ERα, and (4) TrkB agonist therapy decreases the c-caspase-3 only in ERα+/+ female mice hippocampus. Together, these observations provide evidence that female-specific induction of ERα expression confers neuroprotection with TrkB agonist therapy via SFK activation and account for improved functional outcomes in female neonates post-HI.

Cerebrospinal fluid cleaved-tau protein and 9-hydroxyoctadecadienoic acid concentrations in pediatric patients with hydrocephalus

Objective: To ascertain if cerebrospinal fluid cleaved-tau protein and 9-hydroxyoctadecadienoic acid, reflecting potential biomarkers of overall neuronal injury and lipid peroxidation, respectively, are elevated in hydrocephalus patients compared with controls, and if cleaved-tau and 9-hydroxyoctadecadienoic acid levels correlate with each other. Design: Prospective clinical observational study. Setting: Tertiary-care children’s hospital. Patients: Children younger than or equal to 18 yrs who underwent ventriculoperitoneal shunt placement or revision surgery for intrinsic hydrocephalus. Measurements and Main Results: During the study period 12 patients with intrinsic hydrocephalus required ventriculoperitoneal shunt placement or revision. Cerebrospinal fluid cleaved-tau levels were significantly elevated in patients with hydrocephalus (44.7 ± 9.6 ng/mL, n = 11) compared with control patients (0.0 ± 0.0 ng/mL, n = 9; p < 0.0001). Cleaved-tau levels correlated with patient age (r = .609, p = 0.047) and duration of symptoms (r = .755, p = 0.007). No significant difference in cerebrospinal fluid 9-hydroxyoctadecadienoic acid levels between patients with hydrocephalus (24.6 ± 5.7, n = 8) and control patients (24.9 ± 9.3 ng/mL, n = 7) was detected (p = 0.25). There was also no statistically significant correlation between 9-hydroxyoctadecadienoic acid levels and duration of symptoms (r = .668, p = 0.07), nor was there a significant correlation between 9-hydroxyoctadecadienoic acid levels and patient age (r = −.011, p > 0.10). There was no significant relationship between 9-hydroxyoctadecadienoic acid levels and signs of elevated intracranial pressure, nor was there a correlation between 9-hydroxyoctadecadienoic acid levels and cleaved-tau levels. Conclusion: Children with hydrocephalus who have clinical signs of increased intracranial pressure and require ventriculoperitoneal shunt placement or revision exhibit markedly elevated cerebrospinal fluid cleaved-tau levels, suggesting evidence of axonal damage. However, this axonal injury does not seem to be associated with significant lipid peroxidation, at least as assessed by quantifying cerebrospinal fluid 9-hydroxyoctadecadienoic acid at a single, concurrent time point. The significant relationship between age and cerebrospinal fluid cleaved-tau levels suggest that brain injury associated with hydrocephalus may be more pronounced in older children.

Suppression of microglia activation after hypoxia-ischemia results in age-dependent improvements in neurologic injury.

We previously found increased microglial proliferation and pro-inflammatory cytokine release in infant mice compared to juvenile mice after hypoxia-ischemia (HI). The aim of the current study was to assess for differences in the effect of microglial suppression on HI-induced brain injury in infant and juvenile mice. HI was induced in neonatal (P9) and juvenile (P30) mice and minocycline or vehicle was administered at 2h and 24h post-HI. P9 minocycline-treated mice demonstrated early but transient improvements in neurologic injury, while P30 minocycline-treated mice demonstrated sustained improvements in cerebral atrophy and Morris Water Maze performance at 60days post-HI.