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Neonatal encephalopathy due to perinatal hypoxia-ischemia (HI) is a severe condition, and current treatment options are limited. Expression of endogenous osteopontin (OPN), a multifunction glycoprotein, is strongly upregulated in the brain after neonatal HI. Intracerebrally administered OPN has been shown to be neuroprotective following experimental neonatal HI and adult stroke. In the present study, we determined whether intranasal, intraperitoneal or intracerebral treatment with a smaller TAT-OPN peptide is neuroprotective in neonatal mice with HI brain damage. The TAT-OPN peptide exerts bioactivity as it was as potent as full-length OPN in inducing cell adhesion in an in vitro adhesion assay. Intranasal administration of TAT-OPN peptide immediately after HI (T0) or in a repetitive treatment schedule of T0, 3 h, day (D) 1, 2 and 3 after HI did not protect cerebral gray or white matter after HI. Intraperitoneal TAT-OPN treatment at T0 or in two extended treatment schedules (D5, 7, 9, 11, 13, 15 after HI or T0, D1, 3, 5, 7, 9, 11, 13 and 15 after HI) did not result in neuroprotection either. Moreover, no functional improvement (cylinder rearing test and adhesive removal task) was observed following TAT-OPN treatment in any of the intraperitoneal treatment schedules. We validated that the TAT-OPN peptide reached the brain after intranasal or intraperitoneal administration by using an HIV-TAT staining. Finally, also intracerebral administration of the TAT-OPN peptide 1 h after HI did not reduce cerebral damage. Our data show that administration of the TAT-OPN peptide did not exert neuroprotective effects on neonatal HI-induced brain injury or sensorimotor behavioral deficits., (© 2015 S. Karger AG, Basel.)

Antenatal inflammation is associated with increased severity of hypoxic-ischemic (HI) encephalopathy and adverse outcome in human neonates and experimental rodents. We investigated the effect of lipopolysaccharide (LPS) on the timing of HI-induced cerebral tissue loss and gray matter injury, white matter injury and integrity, and the cerebral inflammatory response. On postnatal day 9, mice underwent HI by unilateral carotid artery occlusion followed by systemic hypoxia which resulted in early neuronal damage (MAP2 loss) at 3 h that did not increase up to day 15. LPS injection 14 h before HI (LPS+HI) significantly and gradually aggravated MAP2 loss from 3 h up to day 15, resulting in an acellular cystic lesion. LPS+HI increased white matter damage, reduced myelination in the corpus callosum and increased white matter fiber coherency in the cingulum. The number of oligodendrocytes throughout the lineage (Olig2-positive) was increased whereas more mature myelinating (CNPase-positive) oligodendrocytes were strongly decreased after LPS+HI. LPS+HI induced an increased and prolonged expression of cerebral cytokines/chemokines compared to HI. Additionally, LPS+HI increased macrophage/microglia activation and influx of neutrophils in the brain compared to HI. This study demonstrates the sensitizing effect of LPS on neonatal HI brain injury for an extended time-frame up to 15 days postinsult. LPS before HI induced a gradual increase in gray and white matter deficits, including reduced numbers of more mature myelinating oligodendrocytes and a decrease in white matter integrity. Moreover, LPS+HI prolonged and intensified the cerebral inflammatory response, including cellular infiltration. In conclusion, as the timing of damage and/or involved pathways are changed when HI is preceded by inflammation, experimental therapies might require modifications in the time window, dosage or combinations of therapies for efficacious neuroprotection., (© 2015 S. Karger AG, Basel.)

Objective: To compare neurodevelopmental outcome, mean arterial blood pressure (MABP), and regional cerebral oxygenation (rSco2) between preterm neonates treated for hypotension and controls., Study Design: Preterm neonates (N = 66) with a gestational age (GA) ≤32 weeks, without a patent ductus arteriosus, treated for hypotension (dopamine ≥5 μg/kg/min) were included. Neonates were matched to controls for GA, birth weight, sex, and year of birth. The rSco2 was determined by using near-infrared spectroscopy. Monitoring of MABP, rSco2, and arterial saturation was started at admission and continued for at least 72 hours. Neurodevelopmental outcome was assessed at 18 and 24 months' corrected age by using the Griffiths Mental Development Scales or the Bayley Scales of Infant and Toddler Development, Third Edition., Results: Infants treated for hypotension spent more time with an MABP less than GA (median 9% vs 0%, P < .001) and time with an MABP/rSco2 correlation >0.5 (27% vs 17%, P < .001). Time spent with an rSco2 <50% and neurodevelopmental outcome at 18 and 24 months' corrected age were not significantly different between infants treated for hypotension and controls. The 26 neonates with an rSco2 <50% for >10% of time had a lower neurodevelopmental outcome at 18 months (median 99 vs 104, P = .02)., Conclusion: An MABP less than GA (in weeks) was not associated with lower rSco2 or with lower neurodevelopmental outcome scores. However, regardless of MABP, low rSco2 was associated with lower neurodevelopmental outcome scores. Perfusion/oxygenation variables could be of additional value in neonatal intensive care., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Neonatal encephalopathy is associated with high mortality and life-long developmental consequences. Therapeutic options are very limited. We assessed the effects of D-JNKi, a small peptide c-Jun N-terminal kinase (JNK) MAP kinase inhibitor, on neuroinflammation, mitochondrial integrity and neuronal damage in a neonatal rat model of ischemic brain damage. Hypoxic-ischemic (HI) brain injury was induced in postnatal-day 7 rats by unilateral carotid artery occlusion and hypoxia, and was followed by intraperitoneal D-JNKi treatment. We demonstrate here for the first time that a single intraperitoneal injection with D-JNKi directly after HI strongly reduces neonatal brain damage by >85% with a therapeutic window of at least 6h. D-JNKi treatment also restored cognitive and motor function as analyzed at 9weeks post-insult. Neuroprotective D-JNKi treatment inhibited phosphorylation of nuclear c-Jun (P-c-Jun), and consequently reduced activity of the AP-1 transcription factor and production of cerebral cytokines/chemokines as determined at 3 and 24h post-HI. Inhibition of P-c-Jun by D-JNKi is thought to be mediated via inhibition of the upstream phosphorylation of cytosolic and nuclear JNK and/or by preventing the direct interaction of phosphorylated (P-)JNK with c-Jun. Surprisingly, however, HI did not induce a detectable increase in P-JNK in cytosol or nucleus. Notably, we show here for the first time that HI induces P-JNK only in the mitochondrial fraction, which was completely prevented by D-JNKi treatment. The hypothesis that mitochondrial JNK activation is key to HI brain injury was supported by data showing that treatment of rat pups with SabKIM1 peptide, a specific mitochondrial JNK inhibitor, is also neuroprotective. Inhibition of HI-induced mitochondrial JNK activation was associated with preservation of mitochondrial integrity as evidenced by prevention of ATP loss and inhibition of lipid peroxidation. The HI-induced increase in apoptotic markers (cytochrome c release and caspase 3 activation) as analyzed at 24h post-HI were also strongly reduced by D-JNKi and the mitochondrial anti-apoptotic proteins Bcl-2 and Bcl-xL were upregulated. Neuroprotection was lost after repeated 0+3h D-JNKi treatment which was associated with complete inhibition of the second peak of AP-1 activity and disability to upregulate mitochondrial Bcl-2 and Bcl-xL. We show here for the first time that D-JNKi treatment efficiently protects the neonatal brain against ischemic brain damage and subsequent cognitive and motor impairment. We propose that inhibition of phosphorylation of mitochondrial JNK is a pivotal step in preventing early loss of mitochondrial integrity leading to reduced neuroinflammation and inhibition of apoptotic neuronal loss. Moreover we show the crucial role of upregulation of mitochondrial anti-apoptotic proteins to maintain neuroprotection., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Neonatal encephalopathy induced by perinatal asphyxia is a serious condition associated with high mortality and morbidity. Inflammation after the insult is thought to contribute to brain injury. This inflammatory response to hypoxia-ischemia (HI) may not only occur in the brain but also in peripheral organs. The aim of the present study was to investigate the effect of neonatal HI on the inflammatory response in the liver in comparison to inflammation in the brain., Methods: HI was induced in P7 Wistar rats by unilateral carotid artery occlusion and hypoxia. Cytokine and chemokine mRNA levels were determined in the brain and liver by quantitative PCR. Polarization of brain macrophages to the M1/M2-like phenotype and infiltration of neutrophils were characterized by immunohistochemistry., Results: 3 h after HI, an upregulation of the proinflammatory cytokines TNF-α and IL-1β and anti-inflammatory IL-10 was observed in the ipsilateral hemisphere of the brain compared to mRNA levels in sham-operated animals. Additionally, cerebral CINC-1 and MCP-1 mRNA expressions were increased. We also observed increased numbers of macrophages/microglia of the M1-like phenotype as well as a small increase in granulocyte influx in the ipsilateral hemisphere. Conversely, in the liver 3 h after HI, a downregulation of TNF-α, IL-1β, and MCP-1 and a trend towards an upregulation of IL-10 were observed compared to mRNA levels of sham-operated animals. However, hepatic CINC-1 expression was increased compared to levels in sham-operated animals. Following systemic hypoxia only, no significant changes in the expression of TNF-α, CINC-1 or MCP-1 were observed in the liver compared to sham-operated littermates, except for an upregulation in hepatic IL-1β expression 3 h after hypoxia. Twenty-four hours after insult, cerebral ipsilateral TNF-α, MCP-1 and CINC-1 mRNA expression was still increased, together with an increase in TGF-β expression. Moreover, an increase in macrophages/microglia of the M1-like phenotype was observed together with the appearance of macrophages/microglia of the M2-like phenotype around the cerebral lesion as well as an increase in granulocyte influx in comparison to 3 h after HI. In the liver, 24 h after HI, cytokine and chemokine responses were similar to mRNA levels in sham-operated animals except for a decrease in IL-10 and MCP-1., Conclusion: We describe for the first time that brain damage following neonatal HI induces an early downregulation of the proinflammatory response in the liver. HI induces an early proinflammatory response in the brain with a concomitant increase in influx of neutrophils and polarization of macrophages/microglia to the M1-like phenotype starting at 3 h and increasing up to 24 h after HI. The inflammatory state of the brain changes after 24 h, with an increase in the anti-inflammatory cytokine TGF-β together with the appearance of macrophages/microglia of the M2-like phenotype. The downregulation of proinflammatory cytokines in the liver is not due to systemic hypoxia only, but is induced by the cerebral damage., (Copyright © 2013 S. Karger AG, Basel.)

Background: Preterm infants with hypotension (mean arterial blood pressure [MABP] < gestational age [GA]) are treated with volume expansion and/or dopamine to ensure adequate cerebral perfusion/oxygenation. We used near-infrared spectroscopy to analyze the effects of volume expansion and dopamine on cerebral oxygenation in hypotensive preterm infants without patent ductus arteriosus (PDA)., Patients and Methods: Among 390 infants, 71 (GA < 32 weeks) were hypotensive and eligible for inclusion. Thirty-three infants received volume expansion only (NaCl 0.9%; 20 mL/kg), and 38 received additional dopamine (5 μg/kg per minute). Nine and 11 infants initially treated with dopamine subsequently needed 7.5 and 10 μg/kg per minute, respectively. Seventy-one infants without hypotension were individually matched to serve as controls. MABP, regional cerebral oxygen saturation (rSco(2)), fractional tissue oxygen extraction (cFTOE), and arterial saturation (Sao(2)) were monitored 15 minutes before and 30 and 60 minutes after volume or dopamine and at comparable postnatal ages in controls., Results: No changes in MABP, rSco(2), or cFTOE were found 30 minutes after volume expansion. MABP increased 60 minutes after 5 μg/kg per minute dopamine (median [range]: 28 [19-32] vs 33 [23-46] mm Hg; P < .001). There was a small increase and decrease, respectively, in rSco(2) (63 [43-84] vs 66 [46-87]%; P < .05) and cFTOE (0.33 [0.14-0.56] vs 0.31 [0.07-0.54]1/1; P < .05). However, no differences were found at any time point between controls and infants treated with volume or additional dopamine (5, 7.5, and 10 μg/kg per minute) for rSco(2) or cFTOE., Conclusions: Volume expansion and additional dopamine do not cause any significant change in rSco(2) or cFTOE in hypotensive preterm infants without PDA. We speculate that very preterm infants with hypotension but without signs of a compromised cerebral oxygenation and systemic perfusion might not be in need of antihypotensive therapy.

Background: Long-term clinical trials have shown that daily treatment with recombinant human deoxyribonuclease (rhDNAse) in patients with mild to moderate cystic fibrosis (CF) improves lung function and decreases the number of respiratory exacerbations. The aim of this study was to analyze the effect of rhDNAse on the bacterial colonization of the airways in children with CF., Methods: This was a retrospective cohort study. From the database of the CF Center Utrecht, we selected two groups, an rhDNAse group (daily 2.5 mg rhDNAse) and a control group (no rhDNAse). Primary outcome parameter was the difference in change in bacterial colonization between the treatment and control group during 1.5-year. Secondary outcome parameters were changes in lung function (FEV1) and pulmonary exacerbations., Results: Children treated with rhDNAse showed no significant changes in bacterial colonization during the treatment period, apart from an increase of P. aeruginosa positive cultures, both compared to baseline (53.1% versus 25%, p < 0.05) and control group (no change during study period, 37% versus 37%). The change in FEV1 after one year of treatment was +4.0% in the treatment group versus -0.3% in the control group (p = 0.22). There were no significant changes in number of pulmonary exacerbations., Conclusions: This study showed no significant beneficial decrease in bacterial airway colonization during 1.5-year of treatment with rhDNAse. The positive effects of rhDNAse on the lung function can therefore not be explained by a change in airway colonization.

Background: Airways of cystic fibrosis (CF) patients are colonised with bacteria early in life. We aimed to analyse differences between results of simultaneously taken upper airway (UAW) and lower airway (LAW) cultures, to describe clinical characteristics of patients with positive versus negative cultures and to follow up the patients with P. aeruginosa positive UAW cultures., Methods: Bacteriological and clinical data from 157 children were collected during annual check up. The number of positive UAW and LAW cultures and correspondence between these results and clinical characteristics were analysed., Results: Positive LAW and UAW cultures were found in 79.6% and 43.9% of patients respectively (p<0.001). Patients with positive LAW cultures were significantly older (11.9 vs. 9.8years, p<0.05) and had more LAW symptoms (73.6% vs. 46.7%, p<0.05), especially when P. aeruginosa was found. Patients with positive UAW cultures (especially S. aureus) had more nasal discharge (50.7% vs. 25.0%, p<0.001). In 65% of patients with positive UAW and negative LAW culture for P. aeruginosa the next LAW became P. aeruginosa positive., Conclusion: UAW cultures and LAW cultures differ in children with CF and there are differences in clinical characteristics between patients with positive versus negative culture results. P. aeruginosa positive UAW cultures appeared to precede positive LAW cultures in a substantial part of patients, suggesting some kind of cross-infection between the UAW and LAW., (Copyright (c) 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)