Optimal treatment for women with acute hypertension in pregnancy; a randomized trial comparing intravenous labetalol versus nicardipine.

OBJECTIVES: Blood pressure control in severe hypertension of pregnancy is crucial for mother and neonate. In absence of evidence, guidelines recommend either intravenous labetalol or nicardipine. We compared the effectiveness and safety of these two drugs in women with severe hypertension in pregnancy. STUDY DESIGN: We performed an open label randomized controlled trial. Women with a singleton pregnancy complicated by severe hypertension (systolic ≥ 160 mmHg and/or diastolic ≥ 110 mmHg) requiring intravenous antihypertensive treatment were randomized to intravenous labetalol or intravenous nicardipine. The primary outcome was a composite adverse neonatal outcome defined as severe Respiratory Distress Syndrome (RDS), Broncho Pulmonary Dysplasia (BPD), Intraventricular Hemorrhage (IVH) IIB or worse, Necrotizing Enterocolitis (NEC), or perinatal death defined as fetal death or neonatal death before discharge from the neonatal intensive care unit (NICU). Based on a power analysis, we estimated that 472 women (236 per group) needed to be included to detect a difference of 15% in the primary outcome with 90% power. The study was halted prematurely at 30 inclusions because of slow recruitment and trial fatigue. RESULTS: Between August 2018 and April 2022, we randomized 30 women of which 16 were allocated to intravenous nicardipine and 14 to intravenous labetalol. The composite adverse neonatal outcome was not significantly different between the two groups (25 % versus 43 % OR 0.28 (95 % CI 0.05-1.43), p = 0.12)). Respiratory distress syndrome occurred more often in the labetalol group than in the nicardipine group (42.9 % versus 12.5 %). Neonatal hypoglycemia occurred more often in the nicardipine group than in the labetalol group (31 % versus 7 %). Time until blood pressure control was faster in women treated with nicardipine than in women treated with labetalol (45 (15-150 min vs. 120 (60-127,5) min). CONCLUSION: In our prematurely halted small RCT, we were unable to provide evidence for the optimal choice of treatment for severe hypertension to improve neonatal outcome and/or to obtain faster blood pressure control. Differences in Respiratory distress syndrome and neonatal hypoglycemia between the groups might be the result of coincidental finding due to the small groups included in the study. A larger randomized trial would be needed to determine the safest and most efficacious (intravenous) therapy for severe hypertension in pregnancy. This study emphasizes the challenges of conducting a RCT for the optimal treatment for these women.

Detailed statistical analysis plan for ALBINO: effect of Allopurinol in addition to hypothermia for hypoxic-ischemic Brain Injury on Neurocognitive Outcome - a blinded randomized placebo-controlled parallel group multicenter trial for superiority (phase III).

BACKGROUND: Despite therapeutic hypothermia (TH) and neonatal intensive care, 45-50% of children affected by moderate-to-severe neonatal hypoxic-ischemic encephalopathy (HIE) die or suffer from long-term neurodevelopmental impairment. Additional neuroprotective therapies are sought, besides TH, to further improve the outcome of affected infants. Allopurinol - a xanthine oxidase inhibitor - reduced the production of oxygen radicals and subsequent brain damage in pre-clinical and preliminary human studies of cerebral ischemia and reperfusion, if administered before or early after the insult. This ALBINO trial aims to evaluate the efficacy and safety of allopurinol administered immediately after birth to (near-)term infants with early signs of HIE. METHODS/DESIGN: The ALBINO trial is an investigator-initiated, randomized, placebo-controlled, double-blinded, multi-national parallel group comparison for superiority investigating the effect of allopurinol in (near-)term infants with neonatal HIE. Primary endpoint is long-term outcome determined as survival with neurodevelopmental impairment versus death versus non-impaired survival at 2 years. RESULTS: The primary analysis with three mutually exclusive responses (healthy, death, composite outcome for impairment) will be on the intention-to-treat (ITT) population by a generalized logits model according to Bishop, Fienberg, Holland (Bishop YF, Discrete Multivariate Analysis: Therory and Practice, 1975) and ."will be stratified for the two treatment groups. DISCUSSION: The statistical analysis for the ALBINO study was defined in detail in the study protocol and implemented in this statistical analysis plan published prior to any data analysis. This is in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. TRIAL REGISTRATION: ClinicalTrials.gov NCT03162653. Registered on 22 May 2017.

Feasibility and safety of intranasally administered mesenchymal stromal cells after perinatal arterial ischaemic stroke in the Netherlands (PASSIoN): a first-in-human, open-label intervention study.

BACKGROUND: Perinatal arterial ischaemic stroke (PAIS) is an important cause of neurodevelopmental disabilities. In this first-in-human study, we aimed to assess the feasibility and safety of intranasally delivered bone marrow-derived allogeneic mesenchymal stromal cells (MSCs) to treat PAIS in neonates. METHODS: In this open-label intervention study in collaboration with all neonatal intensive care units in the Netherlands, we included neonates born at full term (≥36 weeks of gestation) with MRI-confirmed PAIS in the middle cerebral artery region. All eligible patients were transferred to the neonatal intensive care unit of the Wilhelmina Children's Hospital. Neonates received one dose of 45-50 × 106 bone-marrow derived MSCs intranasally within 7 days of presenting signs of PAIS. The primary endpoints were acute and subacute safety outcomes, including vital signs, blood markers, and the occurrence of toxicity, adverse events, and serious adverse events. The occurrence of unexpected cerebral abnormalities by a repeat MRI at 3 months of age was a secondary endpoint. As part of standard clinical follow-up at Wilhelmina Children's Hospital, we assessed corticospinal tract development on MRI and performed motor assessments at 4 months of age. This study is registered with ClinicalTrials.gov, NCT03356821. FINDINGS: Between Feb 11, 2020, and April 29, 2021, ten neonates were enrolled in the study. Intranasal administration of MSCs was well tolerated in all ten neonates. No serious adverse events were observed. One adverse event was seen: a mild transient fever of 38°C without the need for clinical intervention. Blood inflammation markers (C-reactive protein, procalcitonin, and leukocyte count) were not significantly different pre-administration versus post-administration and, although thrombocyte levels increased (p=0·011), all were within the physiological range. Follow-up MRI scans did not show unexpected structural cerebral abnormalities. All ten patients had initial pre-Wallerian changes in the corticospinal tracts, but only four (40%) patients showed asymmetrical corticospinal tracts at follow-up MRI. Abnormal early motor assessment was found in three (30%) infants. INTERPRETATION: This first-in-human study demonstrates that intranasal bone marrow-derived MSC administration in neonates after PAIS is feasible and no serious adverse events were observed in patients followed up until 3 months of age. Future large-scale placebo-controlled studies are needed to determine the therapeutic effect of intranasal MSCs for PAIS. FUNDING: Netherlands Organization for Health Research and Development (ZonMw).

Pharmacokinetic/Pharmacodynamic Modelling of Allopurinol, its Active Metabolite Oxypurinol, and Biomarkers Hypoxanthine, Xanthine and Uric Acid in Hypoxic-Ischemic Encephalopathy Neonates.

BACKGROUND: Allopurinol, an xanthine oxidase (XO) inhibitor, is a promising intervention that may provide neuroprotection for neonates with hypoxic-ischemic encephalopathy (HIE). Currently, a double-blind, placebo-controlled study (ALBINO, NCT03162653) is investigating the neuroprotective effect of allopurinol in HIE neonates. OBJECTIVE: The aim of the current study was to establish the pharmacokinetics (PK) of allopurinol and oxypurinol, and the pharmacodynamics (PD) of both compounds on hypoxanthine, xanthine, and uric acid in HIE neonates. The dosage used and the effect of allopurinol in this population, either or not undergoing therapeutic hypothermia (TH), were evaluated. METHODS: Forty-six neonates from the ALBINO study and two historical clinical studies were included. All doses were administered on the first day of life. In the ALBINO study (n = 20), neonates received a first dose of allopurinol 20 mg/kg, and, in the case of TH (n = 13), a second dose of allopurinol 10 mg/kg. In the historical cohorts (n = 26), neonates (all without TH) received two doses of allopurinol 20 mg/kg in total. Allopurinol and oxypurinol population PK, and their effects on inhibiting conversions of hypoxanthine and xanthine to uric acid, were assessed using nonlinear mixed-effects modelling. RESULTS: Allopurinol and oxypurinol PK were described by two sequential one-compartment models with an autoinhibition effect on allopurinol metabolism by oxypurinol. For allopurinol, clearance (CL) was 0.83 L/h (95% confidence interval [CI] 0.62-1.09) and volume of distribution (Vd) was 2.43 L (95% CI 2.25-2.63). For metabolite oxypurinol, CL and Vd relative to a formation fraction (fm) were 0.26 L/h (95% CI 0.23-0.3) and 11 L (95% CI 9.9-12.2), respectively. No difference in allopurinol and oxypurinol CL was found between TH and non-TH patients. The effect of allopurinol and oxypurinol on XO inhibition was described by a turnover model of hypoxanthine with sequential metabolites xanthine and uric acid. The combined allopurinol and oxypurinol concentration at the half-maximal XO inhibition was 0.36 mg/L (95% CI 0.31-0.42). CONCLUSION: The PK and PD of allopurinol, oxypurinol, hypoxanthine, xanthine, and uric acid in neonates with HIE were described. The dosing regimen applied in the ALBINO trial leads to the targeted XO inhibition in neonates treated with or without TH.

Effect of allopurinol in addition to hypothermia treatment in neonates for hypoxic-ischemic brain injury on neurocognitive outcome (ALBINO): study protocol of a blinded randomized placebo-controlled parallel group multicenter trial for superiority (phase III).

BACKGROUND: Perinatal asphyxia and resulting hypoxic-ischemic encephalopathy is a major cause of death and long-term disability in term born neonates. Up to 20,000 infants each year are affected by HIE in Europe and even more in regions with lower level of perinatal care. The only established therapy to improve outcome in these infants is therapeutic hypothermia. Allopurinol is a xanthine oxidase inhibitor that reduces the production of oxygen radicals as superoxide, which contributes to secondary energy failure and apoptosis in neurons and glial cells after reperfusion of hypoxic brain tissue and may further improve outcome if administered in addition to therapeutic hypothermia. METHODS: This study on the effects of ALlopurinol in addition to hypothermia treatment for hypoxic-ischemic Brain Injury on Neurocognitive Outcome (ALBINO), is a European double-blinded randomized placebo-controlled parallel group multicenter trial (Phase III) to evaluate the effect of postnatal allopurinol administered in addition to standard of care (including therapeutic hypothermia if indicated) on the incidence of death and severe neurodevelopmental impairment at 24 months of age in newborns with perinatal hypoxic-ischemic insult and signs of potentially evolving encephalopathy. Allopurinol or placebo will be given in addition to therapeutic hypothermia (where indicated) to infants with a gestational age ≥ 36 weeks and a birth weight ≥ 2500 g, with severe perinatal asphyxia and potentially evolving encephalopathy. The primary endpoint of this study will be death or severe neurodevelopmental impairment versus survival without severe neurodevelopmental impairment at the age of two years. Effects on brain injury by magnetic resonance imaging and cerebral ultrasound, electric brain activity, concentrations of peroxidation products and S100B, will also be studied along with effects on heart function and pharmacokinetics of allopurinol after iv-infusion. DISCUSSION: This trial will provide data to assess the efficacy and safety of early postnatal allopurinol in term infants with evolving hypoxic-ischemic encephalopathy. If proven efficacious and safe, allopurinol could become part of a neuroprotective pharmacological treatment strategy in addition to therapeutic hypothermia in children with perinatal asphyxia. TRIAL REGISTRATION: NCT03162653, www.ClinicalTrials.gov , May 22, 2017.

Neuroprotective strategies following perinatal hypoxia-ischemia: Taking aim at NOS.

Perinatal asphyxia is characterized by oxygen deprivation and lack of perfusion in the perinatal period, leading to hypoxic-ischemic encephalopathy and sequelae such as cerebral palsy, mental retardation, cerebral visual impairment, epilepsy and learning disabilities. On cellular level PA is associated with a decrease in oxygen and glucose leading to ATP depletion and a compromised mitochondrial function. Upon reoxygenation and reperfusion, the renewed availability of oxygen gives rise to not only restoration of cell function, but also to the activation of multiple detrimental biochemical pathways, leading to secondary energy failure and ultimately, cell death. The formation of reactive oxygen species, nitric oxide and peroxynitrite plays a central role in the development of subsequent neurological damage. In this review we give insight into the pathophysiology of perinatal asphyxia, discuss its clinical relevance and summarize current neuroprotective strategies related to therapeutic hypothermia, ischemic postconditioning and pharmacological interventions. The review will also focus on the possible neuroprotective actions and molecular mechanisms of the selective neuronal and inducible nitric oxide synthase inhibitor 2-iminobiotin that may represent a novel therapeutic agent for the treatment of hypoxic-ischemic encephalopathy, both in combination with therapeutic hypothermia in middle- and high-income countries, as well as stand-alone treatment in low-income countries.

Behavioral and neurodevelopmental outcome of children after maternal allopurinol administration during suspected fetal hypoxia: 5-year follow up of the ALLO-trial.

OBJECTIVE: To evaluate the long-term neurodevelopmental and behavioral outcome of antenatal allopurinol treatment during suspected fetal hypoxia. STUDY DESIGN: We studied children born from women who participated in a randomized double-blind placebo controlled multicenter study (ALLO-trial). Labouring women in whom the fetus was suspected to have fetal hypoxia were randomly allocated to receive allopurinol or placebo. At 5 years of age, the children were assessed with 2 parent reported questionnaires, the Ages and Stages Questionnaire (ASQ) and the Child Behavior Checklist (CBCL). A child was marked abnormal for ASQ if it scored below 2 standard deviation under the normative mean of a reference population in at least one domain. For CBCL, a score above the cut-off value (95th percentile for narrowband scale, 85th percentile for broadband scale) in at least one scale was marked as abnormal. RESULTS: We obtained data from 138 out of the original 222 mildly asphyxiated children included in the ALLO-trial (response rate 62%, allopurinol n = 73, placebo n = 65). At 5 years of age, the number of children that scored abnormal on the ASQ were 11 (15.1%) in the allopurinol group versus 11 (9.2%) in the placebo group (relative risk (RR) 1.64, 95% confidence interval (CI): 0.64 to 4.17, p = 0.30). On CBCL 21 children (30.4%) scored abnormal in de allopurinol group versus 12 children (20.0%) in the placebo group (RR 1.52, 95% CI: 0.82 to 2.83, p = 0.18). CONCLUSION: We found no proof that allopurinol administered to labouring women with suspected fetal hypoxia improved long-term developmental and behavioral outcome. These findings are limited due to the fact that the study was potentially underpowered. TRIAL REGISTRATION: NCT00189007 Dutch Trial Register NTR1383.

Repair of neonatal brain injury: bringing stem cell-based therapy into clinical practice.

Hypoxic-ischaemic brain injury is one of most important causes of neonatal mortality and long-term neurological morbidity in infants born at term. At present, only hypothermia in infants with perinatal hypoxic-ischaemic encephalopathy has shown benefit as a neuroprotective strategy. Otherwise, current treatment options for neonatal brain injury mainly focus on controlling (associated) symptoms. Regeneration of the injured neonatal brain with stem cell-based therapies is emerging and experimental results are promising. At present, increasing efforts are made to bring stem cell-based therapies to the clinic. Among all progenitor cell types, mesenchymal stromal (stem) cells seem to be most promising for human use given their neuroregenerative properties and favourable safety profile. This review summarizes the actual state, potential hurdles and possibilities of stem cell-based therapy for neonatal brain injury in the clinical setting. An early version of this paper was presented at the Groningen Early Intervention Meeting which was held in April 2016.

Patent Ductus Arteriosus and Brain Volume.

BACKGROUND AND OBJECTIVES: A hemodynamically significant patent ductus arteriosus (PDA) can compromise perfusion and oxygenation of the preterm brain. Reports suggest that PDA is associated with increased mortality and morbidity. We hypothesize that long-standing low cerebral oxygenation due to PDA might affect brain volume at term equivalent age. METHODS: Observational study in 140 infants investigating the relationship between near-infrared spectroscopy-monitored cerebral oxygen saturation (rSco2) and MRI-assessed regional brain volume and maturation of the posterior limb of the internal capsule at term-equivalent age in 3 groups: those whose PDA closed with indomethacin, those who needed additional surgical closure, and matched controls. RESULTS: The surgery group had the lowest rSco2 values before closure (n = 35), 48% ± 9.7% (mean ± SD) as compared with the indomethacin (n = 35), 59% ± 10.4 (P < .001), and control groups (n = 70), 66% ± 6.9 (P < .001); the highest postnatal age before effective treatment; and the lowest volumes of most brain regions at term-equivalent age. Multiple linear regression analysis showed a significant effect of preductal closure rSco2 on cerebellar volume in this group. No differences were found in maturation of the posterior limb of the internal capsule. CONCLUSIONS: Long-standing suboptimal cerebral oxygenation due to a PDA may negatively influence brain growth, affecting neurodevelopmental outcome.

Assessment of long-term safety and efficacy of intranasal mesenchymal stem cell treatment for neonatal brain injury in the mouse.

BACKGROUND: For clinical translation, we assessed whether intranasal mesenchymal stem cell (MSC) treatment after hypoxia-ischemia (HI) induces neoplasia in the brain or periphery at 14 mo. Furthermore, the long-term effects of MSCs on behavior and lesion size were determined. METHOD: HI was induced in 9-d-old mice. Pups received an intranasal administration of 0.5 × 10(6) MSCs or vehicle at 10 d post-HI. Full macroscopical and microscopical pathological analysis of 39 organs per mouse was performed. Sensorimotor behavior was assessed in the cylinder-rearing test at 10 d, 28 d, 6 mo, and 9 mo. Cognition was measured with the novel object recognition test at 3 and 14 mo post-HI. Lesion size was determined by analyzing mouse-anti-microtubule-associated protein 2 (MAP2) and mouse-anti-myelin basic protein (MBP) staining at 5 wk and 14 mo. RESULTS: At 14 mo post-HI, we did not observe any neoplasia in the nasal turbinates, brain, or other organs of HI mice treated with MSCs. Furthermore, our results show that MSC-induced improvement of sensorimotor and cognitive function is long lasting. In contrast, HI-vehicle mice showed severe behavioral impairment. Recovery of MAP2- and MBP-positive area lasted up to 14 mo following MSC treatment. CONCLUSION: Our results provide strong evidence of the long-term safety and positive effects of MSC treatment following neonatal HI in mice.

Development of cerebral gray and white matter injury and cerebral inflammation over time after inflammatory perinatal asphyxia.

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.

Maternal allopurinol administration during suspected fetal hypoxia: a novel neuroprotective intervention? A multicentre randomised placebo controlled trial.

OBJECTIVE: To determine whether maternal allopurinol treatment during suspected fetal hypoxia would reduce the release of biomarkers associated with neonatal brain damage. DESIGN: A randomised double-blind placebo controlled multicentre trial. PATIENTS: We studied women in labour at term with clinical indices of fetal hypoxia, prompting immediate delivery. SETTING: Delivery rooms of 11 Dutch hospitals. INTERVENTION: When immediate delivery was foreseen based on suspected fetal hypoxia, women were allocated to receive allopurinol 500 mg intravenous (ALLO) or placebo intravenous (CONT). MAIN OUTCOME MEASURES: Primary endpoint was the difference in cord S100ß, a tissue-specific biomarker for brain damage. RESULTS: 222 women were randomised to receive allopurinol (ALLO, n=111) or placebo (CONT, n=111). Cord S100ß was not significantly different between the two groups: 44.5 pg/mL (IQR 20.2-71.4) in the ALLO group versus 54.9 pg/mL (IQR 26.8-94.7) in the CONT group (difference in median -7.69 (95% CI -24.9 to 9.52)). Post hoc subgroup analysis showed a potential treatment effect of allopurinol on the proportion of infants with a cord S100ß value above the 75th percentile in girls (ALLO n=5 (12%) vs CONT n=10 (31%); risk ratio (RR) 0.37 (95% CI 0.14 to 0.99)) but not in boys (ALLO n=18 (32%) vs CONT n=15 (25%); RR 1.4 (95% CI 0.84 to 2.3)). Also, cord neuroketal levels were significantly lower in girls treated with allopurinol as compared with placebo treated girls: 18.0 pg/mL (95% CI 12.1 to 26.9) in the ALLO group versus 32.2 pg/mL (95% CI 22.7 to 45.7) in the CONT group (geometric mean difference -16.4 (95% CI -24.6 to -1.64)). CONCLUSIONS: Maternal treatment with allopurinol during fetal hypoxia did not significantly lower neuronal damage markers in cord blood. Post hoc analysis revealed a potential beneficial treatment effect in girls. TRIAL REGISTRATION NUMBER: NCT00189007, Dutch Trial Register NTR1383.

Neonatal glucocorticoid treatment: long-term effects on the hypothalamus-pituitary-adrenal axis, immune system, and problem behavior in 14-17 year old adolescents.

Neonatal glucocorticoid (GC) treatment is used to prevent bronchopulmonary dysplasia (BPD) in prematurely born babies. In the 1990s, treatment regimens with relatively high doses of dexamethasone (DEX) were common. As an alternative, hydrocortisone (HC) was used. Earlier, we compared long-term effects of both GCs in children aged 7-10 and detected adverse effects of neonatal DEX treatment, but not of HC, on a range of outcomes. The aim of the current cohort study was to investigate whether long-term effects of neonatal DEX were maintained and whether effects of HC remained absent at adolescent age (14-17years). We compared 71 DEX-treated and 67 HC-treated adolescents. In addition, 71 adolescents who were not neonatally treated with GCs participated. All were born <32weeks of gestation. DEX-treated girls showed increased adrenocorticotropic hormone (ACTH) and cortisol responses in the Trier Social Stress Test. The cortisol awakening response was lower in HC-treated participants compared to untreated participants. Negative feedback function of the HPA-axis in the dexamethasone suppression test did not differ between groups. In contrast to our observations at the age of 7-10years, we did not observe group differences in mitogen-induced cytokine production at the age of 14-17years. DEX-treated girls showed more social problems and anxious/depressed behavior than HC-treated girls. Untreated girls showed more problem behavior as well. In conclusion, our results suggest that, especially in girls, neonatal DEX has a programming effect on the HPA-axis and on the ability to adjust to the environment. The loss of group differences on immune system measures indicate that potentially negative effects detected at a younger age subsided.

Intranasal administration of human MSC for ischemic brain injury in the mouse: in vitro and in vivo neuroregenerative functions.

Intranasal treatment with C57BL/6 MSCs reduces lesion volume and improves motor and cognitive behavior in the neonatal hypoxic-ischemic (HI) mouse model. In this study, we investigated the potential of human MSCs (hMSCs) to treat HI brain injury in the neonatal mouse. Assessing the regenerative capacity of hMSCs is crucial for translation of our knowledge to the clinic. We determined the neuroregenerative potential of hMSCs in vitro and in vivo by intranasal administration 10 d post-HI in neonatal mice. HI was induced in P9 mouse pups. 1×10(6) or 2×10(6) hMSCs were administered intranasally 10 d post-HI. Motor behavior and lesion volume were measured 28 d post-HI. The in vitro capacity of hMSCs to induce differentiation of mouse neural stem cell (mNSC) was determined using a transwell co-culture differentiation assay. To determine which chemotactic factors may play a role in mediating migration of MSCs to the lesion, we performed a PCR array on 84 chemotactic factors 10 days following sham-operation, and at 10 and 17 days post-HI. Our results show that 2×10(6) hMSCs decrease lesion volume, improve motor behavior, and reduce scar formation and microglia activity. Moreover, we demonstrate that the differentiation assay reflects the neuroregenerative potential of hMSCs in vivo, as hMSCs induce mNSCs to differentiate into neurons in vitro. We also provide evidence that the chemotactic factor CXCL10 may play an important role in hMSC migration to the lesion site. This is suggested by our finding that CXCL10 is significantly upregulated at 10 days following HI, but not at 17 days after HI, a time when MSCs no longer reach the lesion when given intranasally. The results described in this work also tempt us to contemplate hMSCs not only as a potential treatment option for neonatal encephalopathy, but also for a plethora of degenerative and traumatic injuries of the nervous system.

Antenatal allopurinol reduces hippocampal brain damage after acute birth asphyxia in late gestation fetal sheep.

Free radical-induced reperfusion injury is a recognized cause of brain damage in the newborn after birth asphyxia. The xanthine oxidase inhibitor allopurinol reduces free radical synthesis and crosses the placenta easily. Therefore, allopurinol is a promising therapeutic candidate. This study tested the hypothesis that maternal treatment with allopurinol during fetal asphyxia limits ischemia-reperfusion (I/R) damage to the fetal brain in ovine pregnancy. The I/R challenge was induced by 5 repeated measured compressions of the umbilical cord, each lasting 10 minutes, in chronically instrumented fetal sheep at 0.8 of gestation. Relative to control fetal brains, the I/R challenge induced significant neuronal damage in the fetal hippocampal cornu ammonis zones 3 and 4. Maternal treatment with allopurinol during the I/R challenge restored the fetal neuronal damage toward control scores. Maternal treatment with allopurinol offers potential neuroprotection to the fetal brain in the clinical management of perinatal asphyxia.

Hydrocortisone treatment for bronchopulmonary dysplasia and brain volumes in preterm infants.

OBJECTIVE: To assess whether there was an adverse effect on brain growth after hydrocortisone (HC) treatment for bronchopulmonary dysplasia (BPD) in a large cohort of infants without dexamethasone exposure. STUDY DESIGN: Infants who received HC for BPD between 2005 and 2011 and underwent magnetic resonance imaging at term-equivalent age were included. Control infants born in Geneva (2005-2006) and Utrecht (2007-2011) were matched to the infants treated with HC according to segmentation method, sex, and gestational age. Infants with overt parenchymal pathology were excluded. Multivariable analysis was used to determine if there was a difference in brain volumes between the 2 groups. RESULTS: Seventy-three infants treated with HC and 73 matched controls were included. Mean gestational age was 26.7 weeks, and mean birth weight was 906 g. After correction for gestational age, postmenstrual age at time of scanning, the presence of intraventricular hemorrhage, and birth weight z-score, no differences were found between infants treated with HC and controls in total brain tissue or cerebellar volumes. CONCLUSIONS: In the absence of associated parenchymal brain injury, no reduction in brain tissue or cerebellar volumes could be found at term-equivalent age between infants with or without treatment with HC for BPD.

Intranasal mesenchymal stem cell treatment for neonatal brain damage: long-term cognitive and sensorimotor improvement.

Mesenchymal stem cell (MSC) administration via the intranasal route could become an effective therapy to treat neonatal hypoxic-ischemic (HI) brain damage. We analyzed long-term effects of intranasal MSC treatment on lesion size, sensorimotor and cognitive behavior, and determined the therapeutic window and dose response relationships. Furthermore, the appearance of MSCs at the lesion site in relation to the therapeutic window was examined. Nine-day-old mice were subjected to unilateral carotid artery occlusion and hypoxia. MSCs were administered intranasally at 3, 10 or 17 days after hypoxia-ischemia (HI). Motor, cognitive and histological outcome was investigated. PKH-26 labeled cells were used to localize MSCs in the brain. We identified 0.5 × 10(6) MSCs as the minimal effective dose with a therapeutic window of at least 10 days but less than 17 days post-HI. A single dose was sufficient for a marked beneficial effect. MSCs reach the lesion site within 24 h when given 3 or 10 days after injury. However, no MSCs were detected in the lesion when administered 17 days following HI. We also show for the first time that intranasal MSC treatment after HI improves cognitive function. Improvement of sensorimotor function and histological outcome was maintained until at least 9 weeks post-HI. The capacity of MSCs to reach the lesion site within 24 h after intranasal administration at 10 days but not at 17 days post-HI indicates a therapeutic window of at least 10 days. Our data strongly indicate that intranasal MSC treatment may become a promising non-invasive therapeutic tool to effectively reduce neonatal encephalopathy.

Early life intervention with glucocorticoids has negative effects on motor development and neuropsychological function in 14-17 year-old adolescents.

OBJECTIVE: To reduce the risk of bronchopulmonary dysplasia, preterm infants receive neonatal treatment with glucocorticoids, mostly dexamethasone (DEX). Compared to current protocols, treatment regimens of the late 1980s - early 1990s prescribed high doses of DEX for an extensive period up to 6 weeks. Worldwide at least one million children have been treated with this dose regimen. Previous studies have shown adverse effects of neonatal treatment with the glucocorticoid dexamethasone (DEX) on outcome in children aged 7-10 years. On the other hand, treatment with another glucocorticoid, hydrocortisone (HC), was not related to adverse effects in childhood. In the current study we determined the consequences of early life intervention with DEX or HC in adolescents (age 14-17 years). Besides motor function and intellectual capacities, we also examined fundamental neuropsychological functions which have so far received little attention. METHODS: In an observational cohort study we compared 14-17 year-old adolescents who received DEX (.5 mg/kg/day tapering off to .1 mg/kg/day over 21 days, n=63), or HC (5 mg/kg/day tapering off to 1 mg/kg/day over 22 days, n=67), or did not receive neonatal glucocorticoids (untreated, n=71) after premature birth (gestational age<32 weeks). Because gestational age was shorter and duration of ventilation was longer in the DEX-treated group, all analyses were corrected for these potential confounders. Motor function, IQ, and neuropsychological functions were assessed. RESULTS: DEX-treated group participants scored lower on gross motor skill tasks than their HC-treated and untreated counterparts. A higher proportion of DEX-treated girls needed special education compared to the other groups. DEX-treated adolescents performed poorer on neuropsychological tasks measuring alertness, visuomotor coordination, and emotion recognition. The HC-treated group did not differ from the untreated group. CONCLUSIONS: Even after 14-17 years, neonatal treatment with .5 mg/kg/day DEX was associated with adverse effects on motor function, school level, and neuropsychological functions, whereas treatment with the clinically equally effective dose of 5 mg/kg/day HC was not. Potential physiological mechanisms underlying the differences in dexamethasone and hydrocortisone effects are discussed. Based on the current findings, we recommend early identification of neuropsychological deficits after DEX treatment in order to specify extra educational needs.

Mesenchymal stem cells restore cortical rewiring after neonatal ischemia in mice.

OBJECTIVE: A study was undertaken to investigate the effect of neonatal hypoxic-ischemic (HI) brain damage and mesenchymal stem cell (MSC) treatment on the structure and contralesional connectivity of motor function-related cerebral areas. METHODS: Brain remodeling after HI±MSC treatment in neonatal mice was analyzed using diffusion tensor magnetic resonance imaging, immunohistochemistry, anterograde tracing with biotinylated dextran amine (BDA), and retrograde tracing with fluorescent pseudorabies virus (PRV). RESULTS: MSC treatment after HI reduced contralesional rewiring taking place after HI. Following MSC treatment, fractional anisotropy values, which were increased in both ipsi- and contralesional cortices and decreased in the corpus callosum (CC) after HI, were normalized to the level observed in sham-operated mice. These results were corroborated by myelin basic protein intensity and staining pattern in these areas. Anterograde tracing of ipsilesional motor neurons showed that after MSC treatment, fewer BDA-positive fibers crossed the CC and extended into the contralesional motor cortex compared to HI mice. This remodeling was functional, because retrograde labeling showed increased connectivity between impaired (left) forepaw and the contralesional (left) motor cortex after HI, whereas MSC treatment reduced this connection and increased the connection between the impaired (left) forepaw and the ipsilesional (right) motor cortex. Finally, the extent of contralesional rewiring measured with BDA and PRV tracing was related to sensorimotor dysfunction. INTERPRETATION: This is the first study to describe MSC treatment after neonatal HI markedly reducing contralesional axonal remodeling induced by HI brain injury.

Cell therapy for neonatal hypoxia-ischemia and cerebral palsy.

Perinatal hypoxic-ischemic brain injury remains a major cause of cerebral palsy. Although therapeutic hypothermia is now established to improve recovery from hypoxia-ischemia (HI) at term, many infants continue to survive with disability, and hypothermia has not yet been tested in preterm infants. There is increasing evidence from in vitro and in vivo preclinical studies that stem/progenitor cells may have multiple beneficial effects on outcome after hypoxic-ischemic injury. Stem/progenitor cells have shown great promise in animal studies in decreasing neurological impairment; however, the mechanisms of action of stem cells, and the optimal type, dose, and method of administration remain surprisingly unclear, and some studies have found no benefit. Although cell-based interventions after completion of the majority of secondary cell death appear to have potential to improve functional outcome for neonates after HI, further rigorous testing in translational animal models is required before randomized controlled trials should be considered.