Cytokine dysregulation persists in childhood post Neonatal Encephalopathy.

BACKGROUND: Cytokines are possible mediators of neuroinflammation and associated with adverse outcome in neonatal encephalopathy (NE). Our aim was to explore cytokine response in children with Neonatal Encephalopathy (NE) at school age compared to age-matched controls. METHOD: Follow up at school age, children who had NE and age-matched controls were assessed for their cytokine responses and neurodevelopment outcome. Pro- and anti-inflammatory cytokines in the serum, [Interleukin (IL)-1α, IL-1ß, IL-2, IL-6, IL-8, IL-18, Tumor necrosis factor (TNF)-α, TNF ß, Interferon (IFN)-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), erythropoietin (EPO), IL-10 & IL-1RA] were measured at baseline and in response to in vitro stimulation with lipopolysaccharide (LPS: endotoxin). RESULTS: GM-CSF, TNF-ß, IL-2 IL-6 and IL-8 were significantly elevated at school age following NE (n = 40) compared to controls (n = 37). A rise in GM-CSF, IL-8, TNF-α, IL-1ß, & IL-6 were seen in NE group following LPS stimulation. Relative LPS hypo-responsiveness was also noted in children with severe NE with IL-10, VEGF, EPO and TNF-ß. Elevated TNF-ß was associated with low gross motor scores on assessment at school age. CONCLUSION: School-age children post-NE had significantly altered cytokine responses to endotoxin compared to controls. TNF-ß was associated with adverse developmental outcomes. This suggests the inflammatory process may persist into childhood and a longer therapeutic window may be available for neuroprotection therapies.

Distinct cytokine patterns may regulate the severity of neonatal asphyxia-an observational study.

BACKGROUND: Neuroinflammation and a systemic inflammatory reaction are important features of perinatal asphyxia. Neuroinflammation may have dual aspects being a hindrance, but also a significant help in the recovery of the CNS. We aimed to assess intracellular cytokine levels of T-lymphocytes and plasma cytokine levels in moderate and severe asphyxia in order to identify players of the inflammatory response that may influence patient outcome. METHODS: We analyzed the data of 28 term neonates requiring moderate systemic hypothermia in a single-center observational study. Blood samples were collected between 3 and 6 h of life, at 24 h, 72 h, 1 week, and 1 month of life. Neonates were divided into a moderate (n = 17) and a severe (n = 11) group based on neuroradiological and amplitude-integrated EEG characteristics. Peripheral blood mononuclear cells were assessed with flow cytometry. Cytokine plasma levels were measured using Bioplex immunoassays. Components of the kynurenine pathway were assessed by high-performance liquid chromatography. RESULTS: The prevalence and extravasation of IL-1b + CD4 cells were higher in severe than in moderate asphyxia at 6 h. Based on Receiver operator curve analysis, the assessment of the prevalence of CD4+ IL-1ß+ and CD4+ IL-1ß+ CD49d+ cells at 6 h appears to be able to predict the severity of the insult at an early stage in asphyxia. Intracellular levels of TNF-α in CD4 cells were increased at all time points compared to 6 h in both groups. At 1 month, intracellular levels of TNF-α were higher in the severe group. Plasma IL-6 levels were higher at 1 week in the severe group and decreased by 1 month in the moderate group. Intracellular levels of IL-6 peaked at 24 h in both groups. Intracellular TGF-ß levels were increased from 24 h onwards in the moderate group. CONCLUSIONS: IL-1ß and IL-6 appear to play a key role in the early events of the inflammatory response, while TNF-α seems to be responsible for prolonged neuroinflammation, potentially contributing to a worse outcome. The assessment of the prevalence of CD4+ IL-1ß+ and CD4+ IL-1ß+ CD49d+ cells at 6 h appears to be able to predict the severity of the insult at an early stage in asphyxia.

Cord Blood IL-16 Is Associated with 3-Year Neurodevelopmental Outcomes in Perinatal Asphyxia and Hypoxic-Ischaemic Encephalopathy.

Activation of the inflammatory pathway is increasingly recognized as an important mechanism of injury following neonatal asphyxia and encephalopathy. This process may contribute to the poor prognosis seen in some cases, despite therapeutic hypothermia. Our group has previously identified raised interleukin (IL)-6 and IL-16, measured in umbilical cord blood at birth, to be predictive of grade of hypoxic-ischaemic encephalopathy (HIE). Our aim in this study was to examine the ability of these cytokines to predict the 3-year neurodevelopmental outcome in the same cohort. As part of a prospective, longitudinal cohort study set in a single tertiary maternity unit, term infants with biochemical and clinical evidence of perinatal asphyxia were recruited at birth. Umbilical cord blood was collected and analyzed for IL-6 and IL-16 using a Luminex assay. The neurodevelopmental outcome of these infants was assessed at 3 years using the Bayley Scales of Infant and Toddler Development (Edition 3). Early cord blood measurement of IL-6 and IL-16 and long-term outcome were available in 33/69 infants. Median (IQR) IL-16 differentiated infants with a severely abnormal outcome (n = 6) compared to all others (n = 27), (646 [466-1,085] vs. 383.5 [284-494] pg/mL; p = 0.012). IL-16 levels were able to predict a severe outcome with an area under the receiver-operating characteristic (ROC) curve of 0.827 (95% CI 0.628-1.000; p = 0.014). Levels ≥514 pg/mL predicted a severe outcome with a sensitivity of 83% and a specificity of 81%. IL-16 also outperformed other routine biochemical markers available at birth for the prediction of severe outcome. APGAR scores at 1 and 10 min were also predictive of a severe outcome (p = 0.022 and p = 0.036, respectively). A combination of IL-16 with these clinical markers did not improve predictive value, but IL-16 combined with electroencephalogram grading increased the area under the ROC curve. IL-6 did not show any association with 3-year outcome. This is the first report studying the association of IL-16 measured at birth with long-term outcome in a cohort of neonates with perinatal asphyxia. IL-16 may be an early biomarker of severe injury and aid in the long-term prognostication in infants with HIE.

Fetal asphyctic preconditioning in rats results in a preserved placental inflammatory phenotype at birth.

INTRODUCTION: Perinatal asphyxia (PA) is a major cause of neonatal mortality and morbidity. Research has shown that in rats fetal asphyxia (FA) can provoke neuroprotection against a subsequent more severe perinatal asphyctic insult. This is called fetal asphyctic preconditioning (PC). Our objective was to investigate alterations in the placental inflammatory phenotype associated with PC. METHODS: FA was induced in the rat at embryonic day 17 by reversibly clamping the uterine circulation and PA was induced at birth by submersion of the uterine horns in a saline bath for 19 min. The effect of PC was studied by inducing FA at E17, followed by PA at E21. Placental TNF-α, IL-1ß, IL-6 and IL-10 mRNA and protein levels were measured by qPCR and ELISA. RESULTS: IL-1ß mRNA increased in the labouring FA group, but IL-1ß protein decreased after both FA and PA. In the PC group, IL-1ß mRNA and protein levels were similar to controls. IL-6 protein increased 6 h after FA, however decreased 24 h after FA. IL-6 mRNA was higher in the labouring PA group. IL-10 protein decreased 24 h after FA. At birth, IL-10 mRNA increased in the PA group; however, IL-10 protein decreased in both the PA and the FA group. In the PC group, IL-10 mRNA and protein were similar to control levels. DISCUSSION: Depleted protein concentrations of IL-10 and IL-1ß after one single asphyctic insult were reversed after fetal asphyctic PC. In addition, PC placentas showed less up-regulation of IL-6 mRNA compared to the PA ones. This modulated placental inflammatory phenotype might contribute to the improved neonatal outcome showed after fetal asphyctic PC.

Effect of creatine monohydrate supplementation on relative serum level of IL-6 and IL-18 following neonatal hypoxia ischemia in male albino mouse.

IL-6 has been reported to have neuroprotective effects against cerebral ischemia while IL-8 is a pro inflammatory cytokine structurally related to interleukin-1 family. In the present study, we tried to determine whether 2% Creatine monohydrate supplementation for variable duration influence the IL-6 and 18 concentrations in the serum of male albino mouse following right common carotid artery ligation and hypoxia (8% oxygen) for 25 minutes. Our result revealed that serum concentration of IL6 (P=0.0001) as well as IL-18 (P=0.003) were significantly higher in mice supplemented with creatine monohydrate for 15 weeks than in male albino mice on normal rodent diet following hypoxic ischemic insult indicating that long term creatine monohydrate supplementation up regulates the IL-6 and IL-18 concentrations triggering the neuroinflammatory and neuroprotective responses.

Acute and chronic immunomodulatory changes in rat liver after fetal and perinatal asphyxia.

Hypoxic-ischemic encephalopathy (HIE) caused by fetal and perinatal asphyxia is an important cause of mortality in the neonatal period. Not only will asphyxia affect the brain but also other organs such as the liver and kidneys. Interestingly, it has been shown that liver damage is proportional to the severity of the asphyctic insult, implying an association between liver impairment and HIE. Accordingly, we investigated in an established rat model the acute and chronic hepatic response to both fetal (FA) and perinatal asphyxia (PA). In addition, we assessed whether fetal asphyctic preconditioning (PC) would have any beneficial effect on the liver. Inflammation, ceramide signaling and hepatocellular damage were analyzed in the livers of newborn and adult rats at several short- and long-term time points after both FA and PA. We found that although FA induced an acute inflammatory response, apoptotic mRNA levels and oxidative DNA damage were decreased at 96 h post FA. Whereas increased IL-6 and IL-10 mRNA levels were observed after PA, the combination of FA and PA (PC) attenuated the inflammatory response. Moreover, 6 h after PA anti-apoptotic genes were downregulated and associated with less lipid peroxidation, while preconditioned animals were comparable to controls. In summary, asphyctic PC seems to have an acute protective effect on the liver by modulating the inflammatory, apoptotic and anti-oxidative response. More insight into the hepatic response to asphyxia is necessary, as disturbed hepatic function is associated with metabolic diseases in later life.

Brain inflammation induced by severe asphyxia in newborn pigs and the impact of alternative resuscitation strategies on the newborn central nervous system.

BACKGROUND: We compared the current guidelines for neonatal resuscitation with alternative measures and aimed to find out whether this modulated brain inflammation. METHODS: Progressive asphyxia was induced in 94 newborn pigs until asystole. With the reference being resuscitation guidelines, 30 s of initial positive-pressure ventilation before compression (C) and ventilation (V) (C:V; 3:1) in 21% oxygen, pigs were randomized to (i) ventilation for 30, 60, or 90 s before chest compressions; (ii) C:V ratios of 3:1, 9:3, or 15:2; or (iii) 21% or 100% oxygen. Concentrations of inflammatory markers in the cerebrospinal fluid (CSF) and gene expression in the hippocampus and frontal cortex were measured for different interventions. RESULTS: In CSF, S100 was higher with 90 s than with 30 or 60 s of initial positive-pressure ventilation, whereas concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were higher with 30 than with 60 s. Matrix metalloproteinase-2 (MMP-2) and intracellular adhesion molecule 1 (ICAM-1) were higher with 30 than with 60 s. No other comparison between ratios and oxygen concentrations used yielded significant results. CONCLUSION: With respect to signs of brain inflammation, newly born pigs at asystole should be ventilated for longer than 30 s before chest compressions start. C:V ratios of 9:3 and 15:2 as compared with 3:1, or air instead of pure oxygen, did not modulate inflammatory markers.

The importance of cold-reactive autoantibodies in an asphyxiated infant before therapeutic hypothermia.

Perinatal asphyxia is an important cause of neonatal morbidity and mortality. Hypothermia is an effective treatment of neonatal hypoxic-ischemic encephalopathy in infants. Cold agglutination is a primary or acquired autoimmune disease that involves autoantibodies that lead to hemagglutination at low temperatures lower than that of the body. In this case the importance of cold agglutinins during therapeutic hypothermia is presented.

High cerebrospinal fluid antioxidants and interleukin 8 are protective of hypoxic brain damage in newborns.

The objective was to explain the discrepancy in the development of hypoxic ischemic brain injury (HIE) in some asphyxiated newborns rather than others. Forty newborns were classified according to their cerebrospinal neuron-specific-enolase (CSF-NSE) levels on their 5th-day of life; group 1 with low-NSE (n = 25). The remaining 15 newborns had high-NSE and were further divided into a group with no HIE (n = 10, group 2) and another with HIE (n = 5, group 3). CSF-NSE, total-hydroperoxide (TH), biological-antioxidant-potentials (BAPs), 12 cytokines and erythropoietin (EPO) were measured. The TH/BAP gave the oxidative-stress-index (OSI). The BAPs of serial dilutions of three types of EPO were tested. CSF-NSE and TH and mean OSIs were higher in group 3. IL-8 and mean BAPs were higher in group 2 than in group 1. EPO was less detected in group 3. Serial EPO dilutions correlated with their BAPs. Compensatory antioxidants and IL-8 elevation could be protective of perinatal asphyxic brain injury. Antioxidative effect of EPO could be neuroprotective.

Increased brain levels of F2-isoprostane are an early marker of behavioral sequels in a rat model of global perinatal asphyxia.

Perinatal asphyxia is a major cause of immediate and postponed brain damage in the newborn. It may be responsible for several delayed neurologic disorders and, in this respect, early markers of brain injury would be relevant for therapeutic intervention as well as for identification of infants at high risk for developmental disabilities. Biochemical measurements (brain F2-isoprostane levels) and behavioral tests (ultrasonic vocalization pattern on postnatal days (pnd) 5, 8, and 11, spontaneous motor behaviors on pnd 7 and 12, and homing response on pnd 10) were performed in a rat model of global perinatal asphyxia in the immature neonate. Caesarean section was performed in rats and the pups, still in uterus horns, were placed into a water bath at 37 degrees C for either 10 or 20 min. Caesarean delivered pups were used as controls. Pups experiencing severe (20 min), in contrast to those undergoing the 10 min, asphyctic insult presented with detectable abnormalities including early (two hours after the insult) increase in brain F2-isoprostane (a direct marker of oxidative injury) without detectable changes in PGE2, COX-2 and iNOS levels, and delayed physical (reduced weight gain on pnd 5 and thereafter) and behavioral disturbances (alterations in ultrasound emission on pnd 11 and spontaneous motricity levels mainly). These findings suggest that increased brain F2-isoprostane levels shortly after the asphyctic insult are predictive of delayed behavioral disturbances in the newborn rat. The present 20-min asphyxia model might serve for the assessment of preventive and curative strategies to treat neurologic/behavioral disturbances associated with perinatal asphyxia.

NF-kappaB activation in peripheral blood mononuclear cells in neonatal asphyxia.

Neonatal asphyxia results in hypoxic-ischaemic encephalopathy. Previous studies have demonstrated that brain hypoxia and ischaemia lead to the production of proinflammatory cytokines, including tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-6. Transcription factor NF-kappaB is essential for the expression of these cytokines. We examined whether or not NF-kappaB is activated in peripheral mononuclear cells (PBMC) in neonatal asphyxia by flow cytometry. In addition, we examined the relationship between NF-kappaB activation in PBMC and the neurological prognosis. Flow cytometry analysis demonstrated that the level of NF-kappaB activation in CD14+ monocytes/macrophages of the patients with asphyxia who had neurological sequelae was significantly higher than in the controls, and in the patients with asphyxia who survived (31.7 +/- 7.2%versus 2.5 +/- 0.9%, P = 0.008, and versus 1.6 +/- 1.4%, P = 0.014, respectively). Our findings suggest that NF-kappaB activation in peripheral blood CD14+ monocytes/macrophages in neonatal asphyxia is important for predicting the subsequent neurological sequelae.

[Effect of folium ginkgo extract on the erythrocyte immunity function and serum lipid peroxide in asphyxia neonate].

OBJECTIVE: To observe the changes of erythrocyte immunity and serum lipid peroxide in asphyxia neonate, and to study the effect of Folium Ginkgo extract (FGE) on them. METHODS: Thirty asphyxia neonates were randomly divided into 2 groups, the treated group and the control group, 15 in each group. Erythrocyte C3b receptor rosette rate (E-C3bRR), erythrocyte immune complex rosette rate (E-ICR), blood superoxide dismutase (SOD) activity and serum lipid peroxide (LPO) level were determined at 24 hrs after birth. Conventional treatment was given to both groups and FGE (15 mg/kg.d) was given to the treated group additionally for 7-8 days, then the above-mentioned parameters were re-examined and neonatal behavioral neurological assessment (NBNA) was measured as well. RESULTS: E-C3bRR and SOD lowered, E-ICR and serum LPO increased in the asphyxia neonate significantly (P < 0.05). After treatment, comparison between the two groups showed that E-C3bRR and SOD were higher, E-ICR and serum LPO were lower in the treated group than those in the control group, and NBNA scoring was obviously higher in the former than that in the latter (all P < 0.05). CONCLUSION: Decrease of erythrocyte immunity in asphyxia neonate is related to the declined anti-oxidation ability and lipid peroxidase injury. FGE could suppress the free radical production, scavenge free radicals, antagonize the lipid peroxidation injury of cell membrane and up-regulate erythrocyte immunity. It displays the effects of nerve tissue protection and hypoxia-ischemic brain injury alleviation.

Human perinatal asphyxia: correlation of neonatal cytokines with MRI and outcome.

Clinical studies of the preterm neonate and animal models of asphyxial brain injury both support a role for proinflammatory cytokines in central nervous system (CNS) injury. There are fewer studies of perinatal CNS injury in the full-term neonate. We have performed a prospective cohort study of full-term infants with perinatal asphyxia. Using archived neonatal blood samples, we have analyzed the serum levels of several proinflammatory cytokines. Preliminary results demonstrate an increase in IL-1, IL-6, and TNF-alpha in those children who are deceased at 1 year or who have a diagnosis of cerebral palsy versus those with normal neuromotor outcome. Further analysis will include correlations of cytokine levels with injury on MRI spectroscopy, with neonatal clinical markers of encephalopathy, and with later neurodevelopmental outcome.

Cytokine response in cerebrospinal fluid after birth asphyxia.

Experimental studies suggest that cytokine-mediated inflammatory reactions are important in the cascade leading to hypoxic-ischemic brain injury. The purpose was to study the content of pro- and antiinflammatory cytokines in cerebrospinal fluid (CSF) of asphyxiated and control infants. Samples of CSF were obtained from 20 infants who fulfilled the criteria of birth asphyxia and from seven newborn control subjects. The concentrations of IL-1beta, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, and granulocyte/monocyte colony-stimulating factor (GM-CSF) were determined with ELISA and of IL-6 using a bioassay. The concentration of IL-6 (pg/mL) was higher in asphyxiated (250, 35-543; median, interquartile range) than in control (0, 0-18) infants (p = 0.001). There was also a significant relationship between IL-6 and the degree of HIE, and between IL-6 and outcome. In addition, the content of IL-8 (pg/mL) was higher (p = 0.009) in the asphyxia group (170, 70-1440), than in the the control group (10, 0-30) and there was an association between IL-8 and degree of HIE. The levels of IL-10, TNF-alpha, GM-CSF, and IL-1beta did not differ between groups. In conclusion, the proinflammatory cytokines IL-6 and IL-8 were markedly elevated in CSF of asphyxiated infants, and the intrathecal levels of these cytokines corresponded to the degree of HIE.

Interleukin-6 in the cerebrospinal fluid after perinatal asphyxia is related to early and late neurological manifestations.

OBJECTIVES: To investigate if the concentration of interleukin-6 (IL-6) in the cerebrospinal fluid (CSF) is affected by perinatal asphyxia, and to examine the relation of IL-6 levels in the CSF to the severity of hypoxic-ischemic encephalopathy (HIE), to brain damage, and to the neurological outcome. METHODS: Asphyxiated term neonates were included. Cerebrospinal fluid IL-6 was measured by a sensitive enzyme-linked immunosorbent assay. RESULTS: Twenty neonates were studied: 3 had no HIE, 5 had stage 1, 6 had stage 2, and 6 had stage 3. CSF IL-6 levels (8 to 90 hours of life) were higher in neonates with HIE stage 3 (range, 65 to 2250 pg/mL) when compared with neonates with HIE stage 0 to 2 (<2 pg/mL in 12 neonates, 10 pg/mL in 1). According to neuroimaging techniques and/or pathological examination, 14 neonates were normal, and 5 showed signs of brain damage; 1 was not classified. CSF IL-6 levels were significantly higher in neonates with signs of brain damage. Finally, 5 neonates had adverse outcomes (4 died, 1 had cerebral palsy), 2 had mild motor impairment, and 13 had normal outcomes. CSF IL-6 levels were higher in neonates with adverse outcomes (range, 65 to 2250 pg/mL) compared with neonates with favorable outcomes. CONCLUSION: The magnitude of IL-6 response in the CSF after perinatal asphyxia is related to the severity of neonatal HIE, to brain damage, and to the neurological outcome. Our results suggest that IL-6 might play a role in neonatal hypoxic-ischemic brain damage.

[An immunochemical analysis of the function of the hemato-encephalic barrier in acute fetal hypoxia and asphyxia neonatorum]. / Immunokhimicheskii analiz sostoianiia gematoéntsefalicheskogo bar'era pri ostroi gipoksii plodov i asfiksii novorozhdennykh.

The neurospecific protein alpha 1-globulin has been assayed in serum in order to evaluate the blood-brain barrier in newborns with acute intrapartum hypoxia. The study involved 35 term newborns with birth asphyxia of variable severity. The alpha 1-globulin levels correlated with severity of condition at birth, duration of intrauterine exposure to hypoxia and the presence of obstetric complications and clinical severity of cerebral circulatory disorders. A normal early adaptation and effective therapy reduced serum alpha 1-globulin levels 4-8-fold on the 3rd postnatal day and 6-16-fold on the 5th day. Deterioration of neurological symptoms was parallelled by a significant increase in protein levels (to 6400 ng/ml) at day 5. This evidence may confirm the fact that permeability of the blood-brain barrier is impaired by intrapartum hypoxia.

Phagocytosis of cadmium microcrystals by glass-adherent peripheral blood leucocytes of newborns with various perinatal risks.

Using cadmium microcrystals saturated with human serum albumin as particles for the phagocytic assay, the phagocytic activity of peripheral blood glass-adherent leucocytes of 32 preterm infants, 15 small-for-date newborns, 14 term newborns with perinatal asphyxia and 27 term newborns of diabetic mothers was compared with healthy term newborns and normal adults. A statistically significant increase in both the percent of phagocytosing cells and in the mean number of engulfed particles per cell was found in all groups of "stressed" infants as compared with cells of adults. No difference in the proportion of actively phagocytosing cells was found between term healthy and stressed neonates; as regards the mean number of ingested particles per cell, a slight increase (however, statistically not significant) was found in all groups of infants with perinatal risks compared with normal infants suggesting a temporary stimulation of these cells.