The Importance of Vaccination, Variants and Time Point of SARS-CoV-2 Infection in Pregnancy for Stillbirth and Preterm Birth Risk: An Analysis of the CRONOS Register Study.

Background: The risk of preterm birth (PTB) and stillbirth increases after a SARS-CoV-2 infection during gestation. We aimed to estimate the risk depending on gestational age at infection (early <28 + 0 and late ≥28 weeks of gestation, WoG), virus variants, severity of infection, and vaccination. Methods: PTB was divided into early PTB (<32 + 0) and late PTB (32 + 0-36 + 6 WoG). The prospective register COVID-19 Related Obstetrics and Neonatal Outcome Study (CRONOS) included 8032 pregnant women with a confirmed SARS-CoV-2 infection from 3 April 2020 to 31 December 2022, in Germany and Austria. Results: Stillbirth and early preterm births rates were higher during the Alpha (1.56% and 3.13%) and Delta (1.56% and 3.44%) waves than during the Omicron wave (0.53% and 1.39%). Early SARS-CoV-2 infection increased the risk for stillbirth (aRR 5.76, 95% CI 3.07-10.83) and early PTB before 32 + 0 (aRR, 6.07, 95% CI 3.65-10.09). Hospital admission increased the risks further, especially in the case of ICU admission. Vaccination against SARS-CoV-2 significantly reduced the risk of stillbirth (aRR 0.32, 95% CI 0.16-0.83). Conclusions: This multicentric prospective study shows an increased risk of stillbirth and preterm birth after infection early in pregnancy and therefore the importance of obstetrical surveillance thereafter. Vaccination offers effective protection.

Impact of 2 years of COVID-19 pandemic on preterm birth: Experience from a tertiary center of obstetrics in western Germany.

OBJECTIVE: To compare preterm birth rates and reasons before and during the COVID-19-pandemic using a monocentric, retrospective study. METHODS: Univariate analysis identified differences in rates and reasons for preterm birth and neonatal outcomes between the pre-pandemic period (January 1, 2018 to December 31, 2019) and during the pandemic (January 1, 2020 to December 31, 2021) among all births at our tertiary obstetrical center, the University Hospital of Essen. RESULTS: The cohort consisted of 6086 deliveries with 593 liveborn preterm singletons. During the pandemic, the incidence of preterm birth decreased (10.7% vs. 8.6%; odds ratio [OR] 0.79; 95% confidence interval [CI] 0.66-0.93). Spontaneous preterm birth (43.2% vs. 52.3%; OR 1.47; 95% CI 1.05-2.03), and placenta accreta spectrum disorder (3.7% vs. 8.2%; OR 2.36; 95% CI 1.15-4.84) were more common reasons for preterm birth. Placental dysfunction was a less common reason (34.1% vs. 24.3%; OR 0.62; 95% CI 0.43-0.90). Incidences of preterm premature rupture of membranes (28.13% vs. 40.25%; OR 1.72; 95% CI 1.12-2.43) and oligo-/anhydramnios (3.98% vs. 7.88%; OR 2.06; 95% CI 1.02-4.21) increased. Iatrogenic preterm birth decreased (54.5% vs. 49.5%; OR 0.81; 95% CI 0.58-1.13). Stillbirth rates did not change significantly. Among term births, there were fewer spontaneous deliveries (71.0% vs. 65.8%; OR 0.78; 95% CI 0.69-0.88), and more elective (12.3% vs. 15.1%; OR 1.26; 95% CI 1.07-1.50) and unplanned (9.3% vs. 10.9%; OR 1.19; 95% CI 0.98-1.45) cesarean sections. During the pandemic, more term newborns were admitted to neonatal intensive care (1.4% vs. 2.5%; OR 1.86; 95% CI 1.20-2.88). CONCLUSION: Our results, in line with data from other high-income countries, suggest that the likely reason for the decreased preterm birth rates is the underdiagnosis of pregnancy complications.

Therapeutic Plasma Exchange in Early-Onset Preeclampsia: A 7-Year Monocentric Experience.

Different therapeutic apheresis techniques have been clinically tested to delay preterm delivery in the case of eoPE (early-onset preeclampsia). Our study evaluated the feasibility of TPE (therapeutic plasma exchange) compared to standard-of-care treatment. Twenty patients treated with 95 TPE sessions were included in the final analysis and retrospectively matched with 21 patients with comparable placental dysfunction. Gestational age at admission was 23.75 ± 2.26 versus 27.57 ± 2.68 weeks of gestation (WoG) in the control group (p = < 0.001), mean sFlt-1/PlGF ratio was 1946.26 ± 2301.63 versus 2146.70 ± 3273.63 (p = 0.821) and mean sEng was 87.63 ± 108.2 ng/mL versus 114.48 ± 88.78 ng/mL (p = 0.445). Pregnancy was prolonged for 8.25 ± 5.97 days when TPE was started, compared to 3.14 ± 4.57 days (p = 0.004). The median sFlt-1/PlGF Ratio was 1430 before and 1153 after TPE (-18.02%). Median sEng fell from 55.96 ng/mL to 47.62 mg/mL (-27.73%). The fetal survival rate was higher in TPE-treated cases. NICU (Neonatal Intensive Center Unit) stay was in the median of 63 days in the TPE group versus 48 days in the standard-of-care group (p = 0.248). To date, this monocentric retrospective study, reports the largest experience with extracorporeal treatments in eoPE worldwide. TPE could improve pregnancy duration and reduce sFlt-1 and sEng in maternal serum without impairing neonatal outcomes.

Association of ApoE Genotypes and Recovery From Intracerebral Hemorrhage in Very Low Birth Weight Infants.

BACKGROUND AND PURPOSE: Associations of APOE genotypes with intracerebral hemorrhage (ICH) in preterm infants were previously described. In adults, APOE-ε4 genotype has been proposed as susceptibility factor for impaired recovery after cerebral insult. We here aim to determine APOE genotype-specific neurological consequences of neonatal ICH at school age. METHODS: In this multicenter observational cohort study, very low birth weight (<1500 g, <32 weeks gestational age) children were studied for cerebral palsy (CP) after ultrasound diagnosed ICH stratified by APOE genotype. Follow-up examination was done at the age of 5 to 6 years. Study personnel were blinded for perinatal information and complications. Participants were born between January 1, 2009 and December 31, 2013 and enrolled in the German Neonatal Network. Of 8022 infants primarily enrolled, 2467 children were invited for follow-up between January 1, 2014 and December 31, 2019. Univariate analyses and multivariate logistic regression models were used to assess the impact of APOE genotype (APOE-ε2, APOE-ε3, APOE-ε4) on CP after ICH. RESULTS: Two thousand two hundred fifteen children participated at follow-up, including 363 children with ultrasound diagnosed neonatal ICH. In univariate analyses of children with a history of ICH, APOE-ε3 carriers had lower frequencies of CP (n=33/250; 13.2 [95% CI, 9.4%-17.8%]), as compared to APOE-ε2 (n=15/63; 23.8 [14.6%-35.3%], P=0.037) and -ε4 carriers (n=31/107; 29.0 [21.0%-38.0%], P<0.001), respectively. Regression models revealed an association of APOE-ε4 genotype and CP development (odds ratio, 2.77 [1.44-5.32], P=0.002) after ICH. Notably, at low-grade ICH (grade I) APOE-ε4 expression resulted in an increased rate of CP (n=6/39; 15.4 [6.7-29.0]) in comparison to APOE-ε3 (n=2/105; 1.9 [0.4%-6.0%], P=0.002). CONCLUSIONS: APOE-ε4 carriers have an increased risk for long-term motor deficits after ICH. We assume an effect even after low-grade neonatal ICH, but more data are needed to clarify this issue.

Hypothermia modulates myeloid cell polarization in neonatal hypoxic-ischemic brain injury.

BACKGROUND: Neonatal encephalopathy due to hypoxia-ischemia (HI) is a leading cause of death and disability in term newborns. Therapeutic hypothermia (HT) is the only recommended therapy. However, 30% still suffer from neurological deficits. Inflammation is a major hallmark of HI pathophysiology with myeloid cells being key players, participating either in progression or in resolution of injury-induced inflammation. In the present study, we investigated the impact of HT on the temporal and spatial dynamics of microglia/macrophage polarization after neonatal HI in newborn mice. METHODS: Nine-day-old C57BL/6 mice were exposed to HI through occlusion of the right common carotid artery followed by 1 h hypoxia. Immediately after HI, animals were cooled for 4 h or kept at physiological body core temperature. Analyses were performed at 1, 3 and 7 days post HI. Brain injury, neuronal cell loss, apoptosis and microglia activation were assessed by immunohistochemistry. A broad set of typical genes associated with classical (M1) and alternative (M2) myeloid cell activation was analyzed by real time PCR in ex vivo isolated CD11b+ microglia/macrophages. Purity and composition of isolated cells was determined by flow cytometry. RESULTS: Immediate HT significantly reduced HI-induced brain injury and neuronal loss 7 days post HI, whereas only mild non-significant protection from HI-induced apoptosis and neuronal loss were observed 1 and 3 days after HI. Microglia activation, i.e., Iba-1 immunoreactivity peaked 3 days after HI and was not modulated by HT. However, ex vivo isolated CD11b+ cells revealed a strong upregulation of the majority of M1 but also M2 marker genes at day 1, which was significantly reduced by HT and rapidly declined at day 3. HI induced a significant increase in the frequency of peripheral macrophages in sorted CD11b+ cells at day 1, which deteriorated until day 7 and was significantly decreased by HT. CONCLUSION: Our data demonstrate that HT-induced neuroprotection is preceded by acute suppression of HI-induced upregulation of inflammatory genes in myeloid cells and decreased infiltration of peripheral macrophages, both representing potential important effector mechanisms of HT.

Mutations in TP73 cause impaired mucociliary clearance and lissencephaly.

TP73 belongs to the TP53 family of transcription factors and has therefore been well studied in cancer research. Studies in mice, however, have revealed non-oncogenic activities related to multiciliogenesis. Utilizing whole-exome sequencing analysis in a cohort of individuals with a mucociliary clearance disorder and cortical malformation, we identified homozygous loss-of-function variants in TP73 in seven individuals from five unrelated families. All affected individuals exhibit a chronic airway disease as well as a brain malformation consistent with lissencephaly. We performed high-speed video microscopy, immunofluorescence analyses, and transmission electron microscopy in respiratory epithelial cells after spheroid or air liquid interface culture to analyze ciliary function, ciliary length, and number of multiciliated cells (MCCs). The respiratory epithelial cells studied display reduced ciliary length and basal bodies mislocalized within the cytoplasm. The number of MCCs is severely reduced, consistent with a reduced number of cells expressing the transcription factors crucial for multiciliogenesis (FOXJ1, RFX2). Our data demonstrate that autosomal-recessive deleterious variants in the TP53 family member TP73 cause a mucociliary clearance disorder due to a defect in MCC differentiation.

Incidence Estimates of Perinatal Arterial Ischemic Stroke in Preterm- and Term-Born Infants: A National Capture-Recapture Calculation Corrected Surveillance Study.

INTRODUCTION: Data on valid incidence estimates of perinatal arterial ischemic stroke (PAIS) are scarce. This analysis aims to determine incidence of PAIS in term- and preterm-born infants and to investigate clinical differences related to prematurity. METHODS: This surveillance study (2015-2017) in all German paediatric hospital estimated incidences for MRI-confirmed PAIS in term and preterm infants. To correct for under-reporting, we performed capture-recapture-calculations (CRC) in the most populous federal state and extrapolated nationwide. Differences in clinical presentation in term- and preterm-born infants were assessed. RESULTS: 126 term- and 19 preterm-born infants with PAIS were reported. CRC corrected incidence of PAIS was 22 (95% confidence interval [CI] 17, 27) per 100,000 live births. Stratified by prematurity, the incidence was 32 (95% CI 15, 49) per 100,000 in preterm-born infants and 21 (95% CI 16, 26) per 100,000 term-born infants (significant difference p = 0.001). In symptomatic cases only (n = 120 term born, n = 12 preterm born), incidences did not differ. Risk factor patterns were similar, but number of risk factors in preterm babies was elevated (mean 3.8 vs. 2.9; p = 0.01) and median age at diagnosis was increased (5 vs. 3 days; p = 0.04). Clinical seizures were observed in 88% (106/120) of symptomatic term infants compared to 33% (4/12) in preterm-born infants (p < 0.0001). CONCLUSION: PAIS incidence rates in Germany, extrapolated from estimates for completeness of reporting in the largest federal state, were within the range of other population-based studies. As a novel finding, we detected symptomatic PAIS in preterm-born infants to be as common as in term-born infants although their symptoms were often unspecific.

Clinical Diversity of Cerebral Sinovenous Thrombosis and Arterial Ischaemic Stroke in the Neonate: A Surveillance Study.

INTRODUCTION: Incidence, risk factors, clinical presentation, onset of symptoms, and age at diagnosis differ between neonatal arterial ischaemic stroke (AIS) and cerebral sinovenous thrombosis (CSVT). A more accurate and earlier discrimination of these two entities can be of eminent importance. METHODS: Active surveillance for AIS and CSVT was performed in 345 German paediatric hospitals. Only MRI confirmed cases were included in our analysis. Patients with AIS were compared to CSVT cases with regard to age at diagnosis, pattern of clinical symptoms, and case characteristics. RESULTS: Data on 144 AIS and 51 CSVT neonatal cases were collected from 2015 to 2017. The frequency of reported AIS cases was 2.8 [95% CI 2.1; 3.9] times higher compared to reported CSVT cases. CSVT patients were more likely to be born premature (CSVT 14/48, 29.2%; AIS 19/140, 13.2%; p = 0.02) and to have signs of perinatal acidosis (30.2% CSVT vs. 13.5% AIS; p = 0.01). Generalized seizures and lethargy were more likely to occur in infants with CSVT (p < 0.0001). Age at onset of symptoms and at time of diagnosis were shifted to older ages in CSVT (p < 0.0001). DISCUSSION/CONCLUSION: In the neonatal period, AIS is about three times more common than CSVT. A higher proportion of critically ill infants in CSVT and a later onset of symptoms may indicate that perinatal and postnatal complications are more important for CSVT than for AIS.

Incidence and risk factors of cerebral sinovenous thrombosis in infants.

AIM: To describe the incidence of term and preterm neonatal cerebral sinovenous thrombosis (CSVT) and identify perinatal risk factors. METHOD: This was a national capture-recapture calculation-corrected surveillance and nested case-control study. Infants born preterm and at term with magnetic resonance imaging-confirmed neonatal CSVT were identified by surveillance in all paediatric hospitals in Germany (2015-2017). Incidence was corrected for underreporting using a capture-recapture method in one federal state and then extrapolated nationwide. We reviewed PubMed for comparisons with previously reported incidence estimators. We used a population-based perinatal database for quality assurance to select four controls per case and applied univariate and multivariable regression for risk factor analysis. RESULTS: Fifty-one newborn infants (34 males, 17 females; 14 born preterm) with neonatal CSVT were reported in the 3-year period. The incidence of term and preterm neonatal CSVT was 6.6 (95% confidence interval [CI] 4.4-8.7) per 100 000 live births. Median age at time of confirmation of the diagnosis was 9.95 days (range 0-39d). In the univariate analysis, male sex, preterm birth, hypoxia and related indicators (umbilical artery pH <7.1; 5-minute Apgar score <7; intubation/mask ventilation; perinatal asphyxia), operative vaginal delivery, emergency Caesarean section, and pathological fetal Doppler sonography were associated (p<0.05) with neonatal CSVT. Multivariable regression yielded hypoxia (odds ratio=20.3; 95% CI 8.1-50.8) as the independent risk factor. INTERPRETATION: Incidence of neonatal CSVT was within the range of other population-based studies. The results suggest that hypoxia is an important perinatal risk factor for the aetiology of neonatal CSVT.

Risk factors for perinatal arterial ischaemic stroke: a large case-control study.

AIM: To identify maternal, obstetric, and neonatal risk factors related to perinatal arterial ischaemic stroke (PAIS) diagnosed within 28 days after birth and to understand the underlying pathophysiology. METHOD: For case and control ascertainment, we used active surveillance in 345 paediatric hospitals and a population-based perinatal database for quality assurance of hospital care. We analysed complete cases of PAIS using logistic regression. Multivariate analysis was guided by a directed acyclic graph. RESULTS: After exclusion of records with missing data, we analysed 134 individuals with PAIS and 576 comparison individuals. In univariate analysis, male sex, preterm birth (<37wks gestational age), small for gestational age (SGA), low umbilical artery pH (<7.1), low 5-minute-Apgar score (<7), multiple pregnancies, hypoxia, intubation/mask ventilation, nulliparity, Caesarean section, vaginal-operative delivery, chorioamnionitis, and oligohydramnios were associated with an increased risk. Mutual adjustment yielded male sex (odds ratio [OR] 1.81; 95% confidence interval [CI] 1.20-2.73), multiple birth (OR 3.22; 95% CI 1.21-8.58), chorioamnionitis (OR 9.89; 95% CI 2.88-33.94), preterm birth (OR 1.86; 95% CI 1.01-3.43), and SGA (OR 3.05; 95% CI 1.76-5.28) as independent risk factors. INTERPRETATION: We confirmed the increased risk in males and the role of chorioamnionitis and SGA for PAIS, pointing to the importance of inflammatory processes and fetal-placental insufficiency. Multiple birth and preterm birth were additional risk factors. WHAT THIS PAPER ADDS: Chorioamnionitis and small for gestational age (SGA) precede perinatal arterial ischaemic stroke (PAIS). Chorioamnionitis and SGA are independent risk factors for PAIS. Inflammatory processes and fetal-placental insufficiency are the likely underlying mechanisms. Multiple birth and preterm birth are additional risk factors.

Apolipoprotein E gene polymorphisms and intraventricular haemorrhage in infants born preterm: a large prospective multicentre cohort study.

AIM: Infants born preterm are at risk of intraventricular haemorrhage (IVH) but individual susceptibility related to genes is not well defined in this vulnerable population. Apolipoprotein genotypes APOE2 and APOE4 increase the hazard of cerebral haemorrhages in adults. We investigated whether APOE is associated with prevalence of IVH and is likely to have a particular genotype. METHOD: In this prospective study, 5075 infants born preterm with genotype APOE3 were compared to 965 (APOE2) and 1912 (APOE4) individuals, to analyse the association between APOE genotype and grade III and IV IVH. We used a logistic regression model including gestational age, antenatal steroid treatment, 5-minute Apgar scores less than 3, intubation, pneumothorax, small for gestational age, multiple birth, sex, and maternal descent as independent factors. RESULTS: The APOE2 (20.1%) and APOE4 (19.8%) genotypes were significantly more prevalent in infants with IVH than in those with the APOE3 haplotype (17.4%) (APOE2: odds ratio [OR] 1.33, 95% confidence interval [CI] 1.00-1.76; APOE4: OR 1.39, 95% CI 1.12-1.74). Infants with two polymorphisms had the highest risk of IVH (8.7%; OR 1.63, 95% CI 1.09-2.45). INTERPRETATION: APOE2 and APOE4 genotypes are relevant risk factors for IVH in infants born preterm. Our findings improve our understanding of the genetic contributions to IVH.

Interaction between hypothermia and delayed mesenchymal stem cell therapy in neonatal hypoxic-ischemic brain injury.

Acute hypothermia treatment (HT) is the only clinically established intervention following neonatal hypoxic-ischemic brain injury. However, almost half of all cooled infants still die or suffer from long-lasting neurological impairments. Regenerative therapies, such as mesenchymal stem cells (MSC) appear promising as adjuvant therapy. In the present study, we hypothesized that HT combined with delayed MSC therapy results in augmented protection, improving long-term neurological outcome. Postnatal day 9 (P9) C57BL/6 mice were exposed to hypoxia-ischemia followed by 4 h HT. Murine bone marrow-derived MSC (1 × 106 cells/animal) were administered intranasally at P12. Cytokine and growth factor levels were assessed by ELISA and Luminex® multiplex assay 24 h following MSC delivery. One week after HI, tissue injury and neuroinflammatory responses were determined by immunohistochemistry and western blot. Long-term motor-cognitive outcome was assessed 5 weeks post injury. MSC responses to the brains' environment were evaluated by gene expression analysis in MSC, co-cultured with brain homogenates isolated at P12. Both, MSC and HT improved motor deficits, while cognitive function could only be restored by MSC. Compared to each single therapy, combined treatment led to increased long-lasting motor-cognitive deficits and exacerbated brain injury, accompanied by enhanced endothelial activation and peripheral immune cell infiltration. MSC co-cultured with brain extracts of HT-treated animals revealed increased pro-inflammatory cytokine and decreased growth factor expression. In vivo protein analysis showed higher pro-inflammatory cytokine levels after combined treatment compared to single therapy. Furthermore, HI-induced increase in growth factors was normalized to control levels by HT and MSC single therapy, while the combination induced a further decline below control levels. Our results suggest that alteration of the brains' microenvironment by acute HT modulates MSC function resulting in a pro-inflammatory environment combined with alteration of the homeostatic growth factor milieu in the neonatal hypoxic-ischemic brain. This study delineates potential unexpected side effects of cell-based therapies as add-on therapy for acute hypothermia treatment.

Effects of Poly(ADP-Ribose) Polymerase-1 Inhibition in a Neonatal Rodent Model of Hypoxic-Ischemic Injury.

BACKGROUND: Hypoxia ischemia (HI) to the developing brain occurs in 1-6 in 1000 live births. Large numbers of survivors have neurological long-term sequelae. However, mechanisms of recovery after HI are not understood and preventive measures or clinical treatments are not effective. Poly(ADP-ribose) polymerase-1 is overactivated in response to ischemia. In neonatal mice HI activates PARP-1 but its role in perinatal brain injury remains uncertain. OBJECTIVE: Aim of this study was to explore the effect of TES448 (PARP-1-inhibitor) and hypothermia after an ischemic insult. DESIGN AND METHODS: 10-day-old Wistar rats underwent HI. TES448 was given 10 min, 3 hrs, and 6 hrs after hypoxia. Hypothermia was started 30 min after HI and brains were dissected at P12. Western blotting and histological staining were used to evaluate for degree of injury. RESULTS: Protein expression of PARP-1 levels was diminished after TES448 treatment. Cresyl violet and TUNEL staining revealed decreased injury in male rat pups following TES448 and combined treatment. Female rats showed increased numbers of TUNEL-positive cells after combined therapy. TES448 inhibited microglia activation after hypoxic-ischemic injury. A cellular response including NeuN, Olig2, and MBP was not affected by PARP-1-inhibition. CONCLUSIONS: Inhibition of PARP-1 and hypothermia lead to an alteration of injury but this effect is sexually dimorphic.

Neonatal Arterial Ischemic Stroke - A Hospital Based Active Surveillance Study in Germany.

Background Neonatal arterial ischemic stroke (NAIS) accounts for substantial long term sequelae in children. The potential effectiveness of neuroprotective therapies needs to be evaluated in appropriate studies with sufficient power. Objective To identify annual number of NAIS cases in Germany potentially eligible for randomized interventional trials. Methods Active surveillance for NAIS in 345 pediatric hospitals with questionnaire based validation of reported cases. Results Incidence of NAIS (7.1/100000 births) was in the range of other population-based studies. To design future clinical trials with anticoagulative or regenerative therapies, it is of major importance to distinguish between cases with or without relevant perinatal pathology. Children without underlying disease or premature birth accounted for 56% of all reported NAIS cases (primary NAIS). In 69% of the primary cases clinical seizures were observed. Although 31% showed other, less pathognomonic symptoms, NAIS was diagnosed. Mean time span between onset of symptoms and diagnosis was 2.9 days. The sensitivity of the initial ultrasound performed in all cases was 69%. Conclusions NAIS is a rare but not negligible morbidity in newborns. Asymptomatic children account for 56% of NAIS in all neonates. In these, not only seizures but also other unexplained symptoms should trigger diagnostic work-up with cUS and cMRI. Negative initial ultrasound results do not exclude NAIS.

Sildenafil Enhances Quantity of Immature Neurons and Promotes Functional Recovery in the Developing Ischemic Mouse Brain.

BACKGROUND: Hypoxic-ischemic (HI) injury to the developing brain occurs in 1 out of 1,000 live births and remains a major cause of significant morbidity and mortality. A large number of survivors suffer from long-term sequelae including seizures and neurological deficits. However, the pathophysiological mechanisms of recovery after HI insult are not clearly understood, and preventive measures or clinical treatments are nonexistent or not sufficiently effective in the clinical setting. Sildenafil as a specific phosphodiesterase 5 inhibitor leads to increased levels of the second messenger cyclic guanosine monophosphate (cGMP) and promotes functional recovery and neurogenesis after ischemic injury to the adult brain. OBJECTIVE: Here, we investigated the effect of sildenafil treatment on activation of intracellular signaling pathways, histological and neurogenic response including functional recovery after an ischemic insult to the developing brain. DESIGN/METHODS: Nine-day-old C57BL/6 mice were subjected either to sham operation or underwent ligation of the right common carotid artery followed by hypoxia (8%) for 60 min. Animals were either administered sildenafil (10 mg/kg, i.p.) or vehicle 2 h after hypoxia. A subgroup of animals received multiple injections of 10 mg/kg daily on 5 consecutive days. Pups were either perfusion fixed at postnatal days 14 or 47 for immunohistochemical analysis, or brains were dissected 2, 6, 12, and 24 h after the end of hypoxia and analyzed for cGMP, pAkt, pGSK-3ß, and ß-catenin by means of ELISA or immunoblotting. In addition, behavioral studies using the wire hang test and elevated plus maze were conducted 21 and 38 days after HI injury. RESULTS: Based on cresyl violet staining, single or multiple sildenafil injections did not reveal any differences in injury scoring compared to sham animals. However, cerebral levels of cGMP were altered after sildenafil therapy. Treatment significantly increased numbers of immature neurons, as indicated by doublecortin immunoreactivity in the ipsilateral subventricular zone and striatum. In addition, animals treated with sildenafil after HI insult demonstrated improved functional recovery. pAkt, pGSK-3ß, and ß-catenin levels vary after HI injury but additional sildenafil treatment had no impact on protein expression compared to the level of sham controls. CONCLUSIONS: Here, we report that treatment with sildenafil after HI insult did not improve histological brain injury scores. Nevertheless, our results suggest involvement of the cGMP and PI3K/Akt/GSK-3ß signaling pathway with promotion of a neurogenic response and reduction of neurological deficits. In summary, sildenafil may have a role in promoting recovery from HI injury in the developing brain.

Mesenchymal stem cells attenuate MRI-identifiable injury, protect white matter, and improve long-term functional outcomes after neonatal focal stroke in rats.

Cell therapy has emerged as a potential treatment for many neurodegenerative diseases including stroke and neonatal ischemic brain injury. Delayed intranasal administration of mesenchymal stem cells (MSCs) after experimental hypoxia-ischemia and after a transient middle cerebral artery occlusion (tMCAO) in neonatal rats has shown improvement in long-term functional outcomes, but the effects of MSCs on white matter injury (WMI) are insufficiently understood. In this study we used longitudinal T2-weighted (T2W) and diffusion tensor magnetic resonance imaging (MRI) to characterize chronic injury after tMCAO induced in postnatal day 10 (P10) rats and examined the effects of delayed MSC administration on WMI, axonal coverage, and long-term somatosensory function. We show unilateral injury- and region-dependent changes in diffusion fraction anisotropy 1 and 2 weeks after tMCAO that correspond to accumulation of degraded myelin basic protein, astrocytosis, and decreased axonal coverage. With the use of stringent T2W-based injury criteria at 72 hr after tMCAO to randomize neonatal rats to receive intranasal MSCs or vehicle, we show that a single MSC administration attenuates WMI and enhances somatosensory function 28 days after stroke. A positive correlation was found between MSC-enhanced white matter integrity and functional performance in injured neonatal rats. Collectively, these data indicate that the damage induced by tMCAO progresses over time and is halted by administration of MSCs. © 2016 Wiley Periodicals, Inc.

Effects of PMA (PHORBOL-12-MYRISTATE-13-ACETATE) on the Developing Rodent Brain.

Perinatal infections have a negative impact on brain development. However, the underlying mechanisms leading to neurological impairment are not completely understood and reliable models of inflammation are urgently needed. Using phorbol-myristate-acetate as an activator of inflammation, we investigated the effect on the developing rodent brain. Neonatal rats and mice deficient in IL-18 or IRAK-4 were exposed to PMA. Brains were assessed for regulation of pro- and anti-inflammatory cytokines and cell death 24 hrs, 7 and 14 days after treatment. PMA induced an inflammatory response and caused widespread neurodegeneration in the brains of 3- and 7-day-old rats. In contrast, 14-day-old rats were resistant to the neurotoxic effect of PMA. Histological evaluation at the age of 14 and 21 days revealed a destruction of the cortical microstructure with decreased numerical density of neuronal cells. Mice deficient in IL-18 or IRAK-4 were protected against PMA induced brain injury. PMA treatment during a vulnerable period can alter brain development. IL-18 and IRAK-4 appear to be important for the development of PMA induced injury.

Effects of repetitive exposure to pain and morphine treatment on the neonatal rat brain.

BACKGROUND: Untreated exposure to pain in preterm neonates might damage the vulnerable premature brain and alter development. Pain treatment is limited because analgesic agents may also have adverse neurodevelopmental consequences in newborns. OBJECTIVE: To study the effects of neonatal pain and morphine treatment on the developing brain in a neonatal rat model. METHODS: Newborn rats were randomly assigned to: treatment with formalin injections (group 1), saline injections (group 2) and controls receiving no injections (group 3). Treatment was given on postnatal days 1-3 (model A), 1-5 (model B) and 10-12 (model C). Brains were studied histologically and protein expression was evaluated (protein kinase C epsilon and doublecortin). Effects of preemptive morphine treatment were studied in the same models (models A+M and B+M). RESULTS: Formalin injections resulted in increased apoptotic scores in models A and B. Saline injections increased the number of degenerative cells only in model B. Morphine showed protective effects in formalin-treated animals of model A+M and saline-treated animals of model B+M only. In model C, no neurodegenerative effects were detected. The protein expression of doublecortin showed a pain-related upregulation in the thalamus region, whereas protein kinase C epsilon expression was upregulated in the cortex. CONCLUSIONS: Severe inflammatory pain and pain caused by repetitive injections in neonatal rats may cause major changes in the developing brain during the first week of life. Morphine may only protect the newborn brain against these changes in specific situations.

Magnetic resonance imaging (MRI) as a translational tool for the study of neonatal stroke.

More than half of neonatal stroke survivors have long-term sequelae, including seizures and neurological deficits. Although the immature brain has tremendous potential for recovery, mechanisms governing repair are essentially unexplored. We investigated whether magnetic resonance imaging (MRI) early or late after transient middle cerebral arterial occlusion in postnatal day (P) 10 rats can serve as an intermediate endpoint for long-term studies. Injured animals selected by diffusion-weighted MRI during middle cerebral arterial occlusion were scanned using T2-weighted MRI at P18 and P25 (injury volumes on MRI and histology were compared) or were subjected to contrast-enhanced MRI at P13 to characterize cerebral microcirculatory disturbances and blood-brain barrier leakage. Injury volume during middle cerebral artery occlusion did not predict histological outcome at 2 weeks. Major reductions in injury volume occurred by P18, with no further changes by P25 and correlated with histological injury. Cerebral perfusion was significantly reduced in the injured caudate but blood-brain barrier leakage was small. Therefore, conventional T2-weighted MRI performed during a subchronic injury phase predicts a long-term histological outcome after experimental neonatal focal stroke.

A critical role for Fas/CD-95 dependent signaling pathways in the pathogenesis of hyperoxia-induced brain injury.

OBJECTIVE: Prematurely born infants are at risk for development of neurocognitive impairment in later life. Oxygen treatment has been recently identified as a trigger of neuronal and oligodendrocyte apoptosis in the developing rodent brain. We investigated the role of the Fas death receptor pathway in oxygen-triggered developmental brain injury. METHODS: Six-day-old Wistar rats were exposed to 80% oxygen for various periods (2, 6, 12, 24, 48, and 72 hours), and mice deficient in either Fas (B6.MRL-Tnfrsf6(lpr)) or Fas ligand (B6Smn.C3-Fasl(gld)) and control mice (C57BL/6J) were exposed to 80% oxygen for 24 hours. Polymerase chain reaction, Western blotting, and caspase activity assays of thalamus and cortex tissue were performed. RESULTS: Fas and Fas ligand messenger RNA and protein were upregulated. Furthermore, hyperoxia resulted in induction of downstream signaling events of Fas, such as Fas-associated death domain (FADD), the long and short form of FADD-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein (FLIP-L, FLIP-S), and cleavage of caspase-8 and caspase-3. Injection of a selective caspase-8 inhibitor (TRP801, 1mg/kg) at the beginning of hyperoxia blocked subsequent caspase-3 cleavage in this model. B6.MRL-Tnfrsf6(lpr) mice were protected against oxygen-mediated injury, confirming Fas involvement in hyperoxia-induced cell death. Mice deficient in Fas ligand did not differ from control animals in the amount of cell death. INTERPRETATION: We conclude that neonatal hyperoxia triggers Fas receptor and its downstream signaling events in a Fas ligand-independent fashion. Lack of functional Fas receptors and selective pharmacological inhibition of caspase-8 prevents activation of caspase-3 and provides significant neuroprotection.