A Genome-Wide Analysis of Clinical Chorioamnionitis among Preterm Infants.

OBJECTIVE: To identify single nucleotide polymorphisms (SNPs) associated with clinical chorioamnionitis among preterm infants. STUDY DESIGN: We reanalyzed a genome-wide association study (GWAS) from preterm newborns at less than 30 weeks' gestation. Cases and control definitions were determined using administrative records. There were 213 clinical chorioamnionitis cases and 707 clinically uninfected controls. We compared demographic and clinical outcomes of cases and controls. We performed a GWAS and compared the distribution of SNPs from the background genes and from the immunome genes. We used a Wilcoxon's rank-sum test to compare the SNPs normalized odds ratio and used odds ratios and p-values to determine candidate genes. RESULTS: Infants affected by clinical chorioamnionitis were more likely to have periventricular leukomalacia, high-grade retinopathy, and high-grade intraventricular hemorrhage (IVH). Although a GWAS did not identify SNPs associated with clinical chorioamnionitis at the genome-wide significance level, a direct test on the exonic variants in the human immunome revealed their significant increase of risk in clinical chorioamnionitis. CONCLUSION: Among very preterm infants, clinical chorioamnionitis was associated with periventricular leukomalacia, high-grade retinopathy, and IVH. Our analysis of variants in the human immunome indicates an association with clinical chorioamnionitis in very preterm pregnancies.

Pathogenesis of cerebral palsy through the prism of immune regulation of nervous tissue homeostasis: literature review.

BACKGROUND: The cerebral palsy is highly actual issue of pediatrics, causing significant neurological disability. Though the great progress in the neuroscience has been recently achieved, the pathogenesis of cerebral palsy is still poorly understood. METHODS: In this work, we reviewed available experimental and clinical data concerning the role of immune cells in pathogenesis of cerebral palsy. Maintaining of homeostasis in nervous tissue and its transformation in case of periventricular leukomalacia were analyzed. RESULTS: The reviewed data demonstrate involvement of immune regulatory cells in the formation of nervous tissue imbalance and chronicity of inborn brain damage. The supported opinion, that periventricular leukomalacia is not a static phenomenon, but developing process, encourages our optimism about the possibility of its correction. CONCLUSIONS: The further studies of changes of the nervous and immune systems in cerebral palsy are needed to create fundamentally new directions of the specific therapy and individual schemes of rehabilitation.

Chorioamnionitis in the pathogenesis of brain injury in preterm infants.

Chorioamnionitis (or placental infection) is suspected to be a risk factor for brain injury in premature infants. The suggested association between chorioamnionitis and cystic periventricular leukomalacia and cerebral palsy is uncertain because of the variability of study designs and definitions of chorioamnionitis. Improvements in neonatal intensive care may have attenuated the impact of chorioamnionitis on brain health outcomes. Large multicenter studies using rigorous definitions of chorioamnionitis on placental pathologies and quantitative magnetic resonance techniques may offer the optimal way to clarify the complex role of chorioamnionitis in modifying brain health and long-term outcomes.

Surfactant protein D as a novel therapy for periventricular leukomalacia: is it the missing piece of the puzzle?

Activation of microglia with an inflammatory insult, which plays a central role in periventricular leukomalacia (PVL), results in premyelinating oligodendrocyte death via release of certain cytokines, reactive oxygen and nitrogen species. Toll-like receptor (TLR) 4 is necessary for lipopolysaccharide (LPS) induced oligodenrocyte injury in the CNS. Having an ability to bind TLR 2, 4, and LPS receptor CD14, surfactant protein D (spD) may be a promising agent to counteract the pathways associated with PVL. Supplementation of surfactant treatment with spD may be the key point in prevention of PVL by supression of inflammation and preventing damage to pre-OLs in a vulnerable premature brain operating through TLRs.

Erythropoietin attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain.

Periventricular leukomalacia (PVL), a common neonatal brain white matter (WM) lesion, is frequently associated with cerebral palsy. Growing evidence has indicated that in addition to ischemia/reperfusion injury, cytokine-induced brain injury associated with maternal or fetal infection may also play an important role in the pathogenesis of PVL. Recent studies have shown that administration of lipopolysaccharide (LPS) to pregnant rats causes enhanced expression of the cytokines, i.e., IL-1 beta, TNF-alpha, and IL-6, in fetal brains. In recent years, it has been shown that erythropoietin (EPO) has a critical role in the development, maintenance, protection and repair of the nervous system. In the present study we investigated the effect of EPO on LPS-induced WM injury in Sprague-Dawley rats. LPS (500 microg/kg) suspension in pyrogen-free saline was administered intraperitoneally to pregnant rats at 18 and 19 days of gestation. The control group was treated with pyrogen-free saline. They were given 5,000 U/kg recombinant human EPO. Seven-day-old Sprague-Dawley rat pups were divided into four groups: control group, LPS-treated group, prenatal maternal EPO-treated group (5,000 U/kg, intraperitoneally given to pregnant rats at 18 and 19 days of gestation), and postnatal EPO-treated group (5,000 U/kg, intraperitoneally given to 1-day-old rat pups). Cytokine induction in the postnatal 7-day-old (P7) rat brain after maternal administration of LPS was determined by the ELISA method. The proinflammatory cytokine levels (IL-1 beta, TNF-alpha, and IL-6) in P7 rat pup brains were significantly increased in the LPS-treated group as compared with the control group. Prenatal maternal EPO treatment significantly reduced the concentration of TNF-alpha and IL-6 in the newborn rat brain following LPS injection. The concentration of IL-1 beta was decreased in the intrauterine EPO treatment group. Postnatal EPO treatment significantly decreased only the IL-6 concentration in the newborn rat brain following LPS injection. The concentration of cytokines, IL-1 beta and TNF-alpha, was reduced in the postnatal EPO treatment group. We demonstrated here that LPS administration in pregnant rats at gestational day 18 and 19 induced WM injury in P7 progeny characterized by apoptosis. Prenatal maternal and postnatal EPO treatment significantly reduced the number of apoptotic cells in the periventricular WM. Using immunohistochemistry techniques, we investigated the effects of maternal administration of LPS on myelin basic protein (MBP) staining, as a marker of myelination in the periventricular area in the neonatal rat brain. MBP staining was significantly less and weaker in the brains of the LPS-treated group as compared with the prenatal maternal EPO-treated group. However, the postnatal EPO treatment did not prevent LPS-stimulated loss of MBP-positive staining. In conclusion, especially prenatal maternal EPO treatment attenuates LPS-induced injury by reducing the expression of inflammatory cytokines and sparing MBP in the neonatal rat brain. While the postnatal EPO treatment prevented LPS-induced brain injury this effect was partial. To our knowledge, this is the first study that demonstrates a protective effect of EPO on LPS-induced WM injury in the developing brain. Regarding the wide use of EPO in premature newborns, this agent maybe potentially beneficial in treating LPS-induced brain injury in the perinatal period.

Molecular mechanisms of cell death in periventricular leukomalacia.

OBJECTIVE: To investigate the cytokine-related molecular cascade leading to neural cell death in periventricular leukomalacia (PVL). METHODS: The authors explored potential tumor necrosis factor alpha (TNFalpha) signaling pathways in human brains with PVL and conducted in situ immunohistochemical investigations to search for possible expression of cytokine receptors in these brains. They also investigated likely links to molecules potentially involved in neurocytotoxicity, particularly pathways involving nitrosative-induced apoptosis. RESULTS: TNFalpha overexpression was associated with immune reactivity for p75TNFalphaR2 and p55TNFalphaR1 receptors in affected PVL areas. p75TNFalphaR2 labeling was intense on cerebrovascular endothelial cells in PVL areas, whereas no vascular p55TNFalphaR1 immunoreactivity was detected therein. Immune labeling for both receptors was detected on many white matter parenchymal cells. In contrast, there was no immune reactivity for either receptor in tissues taken from non-PVL areas. Additionally, in situ overexpression of inducible nitric oxide synthase was found in PVL brain regions where apoptotic cell death was detected. CONCLUSIONS: Both p75TNFalphaR2 and p55TNFalphaR1 receptors and nitric oxide may be implicated in the pathogenesis of periventricular leukomalacia.

Population cohort associating chorioamnionitis, cord inflammatory cytokines and neurologic outcome in very preterm, extremely low birth weight infants.

Intrauterine inflammation may relate to neurologic disability among preterm children. We investigated the relationship between chorioamnionitis, cord serum cytokines, and neurologic outcome. Sixty-one consecutively born very preterm extremely low birth weight (ELBW) infants were prospectively enrolled. Histologic inflammation in placenta and umbilical cord and vascular pathology were evaluated. Cord sera were analyzed for five proinflammatory cytokines. Serial brain ultrasound and magnetic resonance imaging were performed for evaluation of intraventricular hemorrhage (IVH grade I-III) and white matter damage (WMD: cystic periventricular leukomalacia or IVH grade IV). Neurologic and neurocognitive outcomes were assessed at the corrected age of 2 y. The incidences of HCA, WMD, and abnormal neurologic outcome were 48%, 13% and 19%, respectively. HCA or high IL-6 in cord serum predicted spontaneous preterm labor with high accuracy. HCA increased the risk of IVH grade II-III. In HCA, without either clinical chorioamnionitis or histologic placental perfusion defect, the children had a low risk of WMD (0%) and a low risk of abnormal neurologic outcome (6%). In HCA, the concentration of IL-6 in cord serum was lower in children with abnormal neurologic outcome than in children with normal neurologic outcome. In HCA and placental perfusion defect (compound defect) the risk of abnormal neurologic outcome was high. Compound placental defect and WMD additively predicted abnormal neurologic outcome. We propose that HCA together with other insults (placental perfusion defect or maternal systemic infection) increases the risk of poor neurologic outcome in very preterm ELBW infants.

Pathophysiology of neonatal brain lesions: lessons from animal models of excitotoxicity.

AIM: The pathophysiology of perinatal brain lesions is probably complex and multifactorial. The development and characterization of distinct yet complementary animal models should help to unravel the cellular and molecular mechanisms underlying perinatal brain lesions. This paper reviews experimental data obtained in animal models of neonatal excitotoxic brain lesions that closely mimic some of the lesions found in human cerebral palsy. CONCLUSION: Available data point to a key role for brain macrophages and oligodendrocytes in neonatal rodent excitotoxic brain lesions and underline the impact of cytokines on these lesions.

Acute lipopolysaccharide-mediated injury in neonatal white matter glia: role of TNF-alpha, IL-1beta, and calcium.

Bacterial infection is implicated in the selective CNS white matter injury associated with cerebral palsy, a common birth disorder. Exposure to the bacterial endotoxin LPS produced death of white matter glial cells in isolated neonatal rat optic nerve (RON) (a model white matter tract), over a 180-min time course. A delayed intracellular Ca(2+) concentration ([Ca(2+)](i)) rise preceded cell death and both events were prevented by removing extracellular Ca(2+). The cytokines TNF-alpha or IL-1beta, but not IL-6, mimicked the cytotoxic effect of LPS, whereas blocking either TNF-alpha with a neutralizing Ab or IL-1 with recombinant antagonist prevented LPS cytotoxicity. Ultrastructural examination showed wide-scale oligodendroglial cell death in LPS-treated rat optic nerves, with preservation of astrocytes and axons. Fluorescently conjugated LPS revealed LPS binding on microglia and astrocytes in neonatal white and gray matter. Astrocyte binding predominated, and was particularly intense around blood vessels. LPS can therefore bind directly to developing white matter astrocytes and microglia to evoke rapid cell death in neighboring oligodendroglia via a calcium- and cytokine-mediated pathway. In addition to direct toxicity, LPS increased the degree of acute cell death evoked by ischemia in a calcium-dependent manner.

Does interleukin-6 genotype influence cerebral injury or developmental progress after preterm birth?

OBJECTIVE: The severity of the proinflammatory response may determine outcome in the critically ill. Genetic variation in the promoter region of the gene encoding the proinflammatory cytokine interleukin-6 (IL-6; -174 CC genotype) may encode enhanced production of IL-6. Our objective was to determine whether the CC genotype is associated with worse early illness severity, neurologic injury, and lower developmental scores among surviving preterm children. METHODS: Genotype was determined from dried blood spots that were taken for neonatal screening tests 7 days or more after birth; outcome was independently assessed as part of a longitudinal study of children of < or =32 weeks' gestational age. RESULTS: CC genotype was associated with worse intensive care indices. Significant hemorrhagic brain injuries occurred in 5 (19%) of 27 children with CC genotype compared with 7 (6%) of 121 children with GC or GG genotype, and images consistent with white matter damage (ventriculomegaly or cystic periventricular leukomalacia) occurred in 9 (26%) of CC patients compared with 9 (7%) in GC/GG children. Disability occurred significantly more often in CC children: 8 (31%) compared with 16 (13%). A similar trend was also noted in children with cerebral palsy (15% compared with 7%, respectively). Developmental, cognitive, and motor scores at 2 years and 5.5 years were independent of genotype among children with or without disability. CONCLUSIONS: In a population of surviving children who were born at < or =32 weeks' gestational age, variation of the gene that may increase IL-6 synthesis is associated with disabling brain injury but not cognitive development despite association with worse early critical care indices.

Periventricular leukomalacia following neonatal and infant cardiac surgery.

The dramatic reduction in surgical mortality following repair of congenital heart defects has been accompanied by increasing recognition of adverse neurodevelopmental sequelae in some children. Neurodevelopmental abnormalities following neonatal and infant cardiac surgery include mild cognitive impairment, expressive speech and language abnormalities, impaired attention and executive function, impaired visual and spatial motor skills, as well as learning disabilities. These defects result in a significant need for early intervention, as well as rehabilitative and special education services. Central nervous system injury following repair of congenital heart defects results from a complex interaction of patient specific and environmental factors. Recent studies suggest that cerebral white matter injury characterized by periventricular leukomalacia (PVL) is common in infants with congenital heart disease particularly following cardiac surgery. Studies at our institution show that this occurs in greater than 50% of neonates following cardiac surgery, but is rare in older infants. Prolonged exposure to cardiopulmonary bypass (with or without deep hypothermic circulatory arrest) is a risk factor for PVL, possibly secondary to the systemic inflammatory response to cardiopulmonary bypass. Hypotension and hypoxemia in the early postoperative period, especially diastolic hypotension, significantly increase the risk of PVL. Future studies are needed to determine significance of PVL as a marker for long-term developmental dysfunction.

Immune mechanisms in the pathogenesis of cerebral palsy: implication of proinflammatory cytokines and T lymphocytes.

Beyond the already known aetiopathogenic factors linked to periventricular leukomalacia, a major pathological substrate in cerebral palsy, immune-inflammatory mechanisms have recently been implicated. Thus proinflammatory cytokines have been shown to be involved very early in the course of this disorder. Additionally, markers of T-lymphocyte activation were upregulated. These findings provide new insight into mechanisms underlying neural cell death in this condition and might open new horizons for preventive and therapeutic strategies.

Possible correlation between high levels of IL-18 in the cord blood of pre-term infants and neonatal development of periventricular leukomalacia and cerebral palsy.

Periventricular leukomalacia (PVL) is a neonatal white matter damage of the brain of pre-term infants that often leads to cerebral palsy (CP). At present, diagnosis of PVL can be done by magnetic resonance imaging (MRI) and ultrasonography only when the infant is at least one week of age. No biochemical methods are available to identify high-risk infants at birth. Cytokines are usually not present in the cord blood but recently an elevation of IL-6 and TNF-alpha levels has been reported in amniotic fluid, cord blood and brain sections of infants with white matter damages. Levels of interleukin-18 (IL-18), a pleiotropic cytokines expressed in the brain and many other tissues, are highly sensitive to pathophysiological changes to raise the possibility that IL-18 may provide a useful indicator of PVL. The cord blood from 17 pre-term infants with PVL, 38 pre-term infants without PVL, and 30 normal full-term infants were retrospectively analysed for IL-18, IL-6, IL-1beta, and TNF-alpha. The possible factors involved in alteration of IL-18 concentration in relation to PVL and CP were examined. IL-18 is undetectable in the cord blood of normal full-term infants. However, high levels of IL-18 exist in the cord blood samples obtained from pre-term infants who neonatally developed PVL followed by CP. For pre-term infants under 35 weeks of gestation, seven out of eight showing more than 200 pg/ml of IL-18 (87.5%) developed PVL neonatally, with five of them subsequently developing CP. In contrast, only five out of 38 pre-term infants with less than 100 pg/ml of IL-18 (13.2%) developed PVL. For pre-term infants with less than 30 weeks of gestation, eight out of nine showing more than 100 pg/ml of IL-18 (88.9%) developed PVL, with six of these eight (75%) developing CP later. In conclusion, the presence of high levels of IL-18 in the cord blood of the pre-term infants is correlated with the incidence of PVL and CP and may provide a prognostic marker applicable at birth.

Inflammatory cytokines in the pathogenesis of periventricular leukomalacia.

BACKGROUND: Periventricular leukomalacia (PVL) affects the developing white matter of neonatal brain. Inflammatory and infectious conditions are implicated in the cause of PVL. METHODS: The authors investigated the in situ expression of proinflammatory cytokines (interleukin-1beta and -6, tumor necrosis factor alpha [TNFalpha]), adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1) and inflammatory cell markers (CD68, leukocyte common antigen, human leukocyte antigen II) in 19 neonatal brains with PVL. The authors compared the findings with matched non-PVL brains. RESULTS: The inflammatory reaction detected at the early stage of PVL extends until the latest phase of cystic cavitation, though at an attenuated level. There is high expression of TNFalpha and to a lesser extent interleukin-1beta; interleukin-6 remains undetectable. Cytokine immunoreactivity is detected in PVL cases both with and without infection. However, cytokine production was higher with infection. A different pattern of cytokine expression was observed in anoxic brains without PVL: TNFalpha immunoreactivity was significantly lower than the PVL group. CONCLUSIONS: An immune-mediated inflammatory process may play a role in PVL. TNFalpha, a myelinotoxic factor, may be the major mediator.

New research directions in neuroepidemiology.

Many of the risk factors previously identified for disorders such as Alzheimer's disease, periventricular leukomalacia, multiple sclerosis, stroke, cerebral palsy, mental retardation, and acquired learning and attention disorders ultimately may be shown to damage the central and peripheral nervous systems through activation of inflammatory mediators. The challenge to epidemiologists in the future is to expand use of epidemiologic methods to explore how immune-mediated insults produce CNS disorders in human populations. Studies of the association of use of nonsteroidal anti-inflammatory drugs with risk of Alzheimer's disease and those of the association of immune parameters with risk of cerebral palsy are excellent examples of how epidemiology can contribute to our understanding of the causes of neurologic and/or neurodevelopmental disorders. Many of the immune parameters of interest have short half-lives and are difficult to measure outside of the laboratory setting. Questions also remain as to the proper timing of measurements in relation to the initial insult and, in some cases, which tissue is the most appropriate to sample. These measurement issues will need to be resolved before use of immune biomarkers in epidemiologic studies of the etiologies of neurologic disorders can be fully realized. Epidemiologists are most likely to help identify ways to prevent neurologic disorders if they are knowledgeable about the molecular biology of inflammation, modulators of CNS vulnerability, and genetic polymorphisms that influence both inflammation and CNS vulnerability and are prepared to become adept at biomarker epidemiology. This does not necessarily compel them to gain extensive knowledge of neurobiology. Rather, neuroepidemiology in the 21st century will require increased collaboration between epidemiologists, neurologists, and neurobiologists.