Temporal patterns of circulating cell-free DNA (cfDNA) in a newborn piglet model of perinatal asphyxia.

Perinatal asphyxia is a severe medical condition resulting from oxygen deficiency (hypoxia) at the time of birth, causing worldwide approximately 680,000 newborn deaths every year. Better prediction of severity of damages including early biomarkers is highly demanded. Elevated levels of circulating cell-free DNA (cfDNA) in blood have been reported for a range of different diseases and conditions, including cancer and prematurity. The objective of this study was to validate methods for assessing cfDNA in blood and cerebrospinal fluid (CSF) and to explore temporal variations in a piglet model of neonatal hypoxia-reoxygenation. Different cfDNA extraction methods in combination with cfDNA detection systems were tested, including a fluorescent assay using SYBR Gold and a qRT-PCR-based technique. Newborn piglets (n = 55) were exposed to hypoxia-reoxygenation, hypoxia-reoxygenation and hypothermia, or were part of the sham-operated control group. Blood was sampled at baseline and at post-intervention, further at 30, 270, and 570 minutes after the end of hypoxia. Applying the fluorescent method, cfDNA concentration in piglets exposed to hypoxia (n = 32) increased from 36.8±27.6 ng/ml prior to hypoxia to a peak level of 61.5±54.9 ng/ml after the intervention and deceased to 32.3±19.1 ng/ml at 570 minutes of reoxygenation, whereas the group of sham-operated control animals (n = 11) revealed a balanced cfDNA profile. Animals exposed to hypoxia and additionally treated with hypothermia (n = 12) expressed a cfDNA concentration of 54.4±16.9 ng/ml at baseline, 39.2±26.9 ng/ml at the end of hypoxia, and of 41.1±34.2 ng/ml at 570 minutes post-intervention. Concentrations of cfDNA in the CSF of piglets exposed to hypoxia revealed at post-intervention higher levels in comparison to the controls. However, these observations were only tendencies and not significant. In a first methodological proof-of-principle study exploring cfDNA using a piglet model of hypoxia-reoxygenation variations in the temporal patterns suggest that cfDNA might be an early indicator for damages caused by perinatal asphyxia.

Fas-ligand and interleukin-6 in the cerebrospinal fluid are early predictors of hypoxic-ischemic encephalopathy and long-term outcomes after birth asphyxia in term infants.

BACKGROUND: Cerebral ischemia generates neuroinflammation that can induce neural cell death. This cohort study assessed whether Fas-ligand (FasL) and interleukin (IL)-6 levels in the cerebrospinal fluid (CSF) after hypoxic-ischemic encephalopathy (HIE) can serve as biomarkers of hypoxic brain injury in neonates. METHODS: Term infants (> 37-week gestational age) who were admitted to the neonatal intensive care unit of Karolinska University Hospital in years 2002 to 2004 with perinatal asphyxia were enrolled prospectively. Control infants without brain pathology underwent lumbar puncture for suspected infection. FasL and IL-6 levels were measured in the CSF, by enzyme-linked immunosorbent assays. All patients underwent neurological assessment at 18 months. HIE was classified as mild, moderate, or severe (HIE I-III). Adverse neurological outcome at 18 months was defined as a mental developmental index < 85, deafness, blindness, cerebral palsy, or seizure disorder. RESULTS: Of the 44 HIE patients, 14, 16, and 14 had HIE-I, HIE-II, and HIE-III, respectively. HIE-II and HIE-III patients had higher FasL and IL-6 levels than HIE-I patients and the 20 controls (all p < 0.0001). Patients with adverse outcomes had higher FasL and IL-6 levels than patients with normal outcomes and controls (both p < 0.0001). On receiver-operator curve analyses, FasL and IL-6 (alone and together) were highly predictive of HIE grade and outcome (areas under the curve range 0.86-0.94) and showed high sensitivity (66.7-100%). These biomarkers performed better than cord blood pH (areas under the curve: HIE grade = 0.80, adverse outcomes = 0.86). CONCLUSION: CSF biomarkers FasL and IL-6 predicted severity of encephalopathy and long-term outcomes in post-asphyxiated infants better than a standard biomarker.

A comparison of blood and cerebrospinal fluid cytokines (IL-1ß, IL-6, IL-18, TNF-α) in neonates with perinatal hypoxia.

Perinatal hypoxia-ischemia is a specific and important pathological event in neonatal care practice. The data on relationship between the concentrations of cytokines in blood and cerebrospinal fluid (CSF) and perinatal brain injury are scarce. The aim of this study is to evaluate changes in interleukin (IL-1ß, IL-6, and IL-18) and tumor necrosis factor alpha (TNF-α) levels in newborns with perinatal hypoxia (PNH). CSF and serum samples of 35 term and near-term (35-40 weeks) newborns with PNH, at the age of 3-96 hours, were analyzed using enzyme-linked immunosorbent assay. Control group consisted of 25 non-asphyxic/non-hypoxic infants of the same age sampled for clinically suspected perinatal meningitis, but proven negative and healthy otherwise. The cytokine values in CSF and serum samples were determined in relation to initial hypoxic-ischemic encephalopathy (HIE) staged according the Sarnat/Sarnat method, and compared with neurological outcome at 12 months of age estimated using Amiel-Tison procedure. The concentrations of IL-6 and TNF-α in serum of PNH patients were significantly higher compared to control group (p = 0.0407 and p = 0.023, respectively). No significant difference between average values of cytokines in relation to the stage of HIE was observed. Significantly higher levels of IL-6 and IL-18 corresponded to a mildly abnormal neurological outcome, while higher levels of IL-6 and TNF-α corresponded to a severely abnormal neurological outcome, at 12 months of age. Elevated serum levels of IL-6 and TNF-α better corresponded with hypoxia/ischemia compared to CSF values, within 96 hours of birth. Also, higher serum levels of IL-6, TNF-α, and IL-18 corresponded better with abnormal neurological outcome at 12 months of age, compared to CSF values.

The potentials and limitations of neuro-biomarkers as predictors of outcome in neonates with birth asphyxia.

Perinatal asphyxia and its complication, hypoxic-ischemic encephalopathy, are still among the major causes of perinatal mortality and morbidity. Despite accurate standard postnatal monitoring procedures, the post-insult period is crucial because at a time when radiologic pictures are still silent, brain damage may already be at a subclinical stage. Against this background, the measurement of quantitative parameters, such as constituents of nervous tissue, that are able to detect subclinical lesions at a stage when routine brain monitoring procedures are still silent, could be particularly useful. Therefore, in the present review we report the potentials and limitations of biomarkers in predicting outcome in neonates complicated by perinatal asphyxia.

Perinatal Asphyxia May Influence the Level of Beta-Amyloid (1-42) in Cerebrospinal Fluid: An Experimental Study on Newborn Pigs.

OBJECTIVE: Total tau (T-tau), phosphorylated tau (p-Tau) and Beta-Amyloid 1-42 (AB42) in Cerebrospinal Fluid (CSF) are useful biomarkers in neurodegenerative diseases. The aim of the study was to investigate the role of these and other CSF biomarkers (T-tau, p-Tau, AB42, S100B and NSE), during hypoxia-reoxygenation in a newborn pig model. DESIGN: Thirty newborn pigs were included in a study of moderate or severe hypoxia. The moderate hypoxia group (n = 12) was exposed to global hypoxia (8% O2) until Base excess (BE) reached -15 mmol/l. The pigs in the group exposed to severe hypoxia (n = 12) received 8% O2 until BE reached -20 mmol/l or mean Blood Pressure fell below 20 mm Hg, The control group (n = 6) was kept at room air. For all treatments, the CSF was collected at 9.5 hours after the intervention. RESULTS: The level of AB42 in CSF was significantly lower in the pigs exposed to severe hypoxia compared with the control group, 922(SD +/-445)pg/ml versus. 1290(SD +/-143) pg/ml (p<0.05), respectively. Further, a non-significant reduction of AB42 was observed in the group exposed to moderate hypoxia T-tau and p-Tau revealed no significant differences between the intervention groups and the control group, however a significantly higher level of S100B was seen in the CSF of pigs receiving hypoxia in comparison to the level in the control group. Further on, there was a moderate negative correlation between the levels of AB42 and S100B in CSF, as well as a moderate negative correlation between Lactate in blood at end of hypoxia and AB42 in CSF. INTERPRETATION: This is the first study to our knowledge that demonstrated a significant drop in AB42 in CSF after neonatal hypoxia. Whether or not this has an etiological basis for adult neurodegenerative disorders needs to be studied with additional experiments and epidemiological studies. AB42 and S100B are significantly changed in neonatal pigs subjected to hypoxia compared to controls and thus may be valuable biomarkers of perinatal asphyxia.

PGE-metabolite levels in CSF correlate to HIE score and outcome after perinatal asphyxia.

AIM: Acute anoxic exposure rapidly increases prostaglandin E2 (PGE2 ) production and release in neonatal mice brains. We hypothesize that PGE2 is released in human cerebrospinal fluid (CSF) during perinatal asphyxia and that it might be used as a biomarker for perinatal asphyxia. METHODS: In full-term infants with lumbar puncture performed within 72 h of birth (n = 35), CSF was analysed for prostaglandin E2 metabolite (PGEM) using an enzyme immunoassay. Term infants with suspected but unverified infections were used as controls (n = 11). Hypoxic-ischaemic encephalopathy (HIE) was classified as mild, moderate or severe (HIE I-III). Neurological assessment of surviving patients was performed at 18 months of age. RESULTS: Prostaglandin E2 metabolite levels correlated to a low Apgar score at 5 min (p < 0.01) and 10 min (p < 0.01), a low pH (p < 0.001) and HIE score (p < 0.05). The HIE-III cases (n = 7) had significantly higher PGEM levels compared with both controls and the HIE-I group (n = 8). Irrespective of HIE grade, patients with adverse or fatal outcome had higher PGEM values compared with controls and asphyxiated infants with normal outcome (p < 0.05). CONCLUSIONS: PGE2 is released during anoxic events in newborn infants, and PGEM may be useful as a biomarker for estimating degree of insult and predicting long-term outcome after perinatal asphyxia.

Microglia/macrophage-derived inflammatory mediators galectin-3 and quinolinic acid are elevated in cerebrospinal fluid from newborn infants after birth asphyxia.

Activation of microglia/macrophages is important in neonatal hypoxic-ischemic (HI) brain injury. Based on experimental studies, we identified macrophage/microglia-derived mediators with potential neurotoxic effects after neonatal HI and examined them in cerebrospinal fluid (CSF) from newborn infants after birth asphyxia. Galectin-3 is a novel inflammatory mediator produced by microglia/macrophages. Galectin-3 is chemotactic for inflammatory cells and activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase resulting in production and release of reactive oxygen species (ROS). Matrix metalloproteinase-9 (MMP-9) is a tissue-degrading protease expressed by activated microglia in the immature brain after HI. Both galectin-3 and MMP-9 contribute to brain injury in animal models for neonatal HI. Quinolinic acid (QUIN) is a neurotoxic N-methyl-D-aspartate (NMDA) receptor agonist also produced by activated microglia/macrophages. Galectin-3 and MMP-9 were measured by ELISA and QUIN by mass spectrometry. Asphyxiated infants (n=20) had higher levels of galectin-3 (mean (SEM) 2.64 (0.43) ng/mL) and QUIN (335.42 (58.9) nM) than controls (n=15) (1.36 (0.46) ng/mL and 116.56 (16.46) nM, respectively), p<0.05 and p<0.01. Infants with septic infections (n=10) did not differ from controls. Asphyxiated infants with abnormal outcome had higher levels of galectin-3 (3.96 (0.67) ng/mL) than those with normal outcome (1.76 (0.32) ng/mL), p=0.02, and the difference remained significant in the clinically relevant group of infants with moderate encephalopathy. MMP-9 was detected in few infants with no difference between groups. The potentially neurotoxic macrophage/microglia-derived mediators galectin-3 and QUIN are increased in CSF after birth asphyxia and could serve as markers and may contribute to injury.

Homovanillic acid in cerebrospinal fluid of 1388 children with neurological disorders.

AIM: To determine the prevalence of dopaminergic abnormalities in 1388 children with neurological disorders, and to analyse their clinical, neuroradiological, and electrophysiological characteristics. METHOD: We studied biogenic amines in 1388 cerebrospinal fluid (CSF) samples from children with neurological disorders (mean age 3y 10mo, SD 4y 5mo; 712 males, 676 females. Correlations among CSF homovanillic acid (HVA) values and other biochemical, clinical, neuroradiological, and electrophysiological parameters were analysed. RESULTS: Twenty-one patients with primary dopaminergic deficiencies were identified. Of the whole sample, 20% showed altered HVA. We report neurological diseases with abnormal CSF HVA values such as pontocerebellar hypoplasia, perinatal asphyxia, central nervous system infections, mitochondrial disorders, and other genetic diseases. Overlapping HVA levels between primary and secondary dopamine deficiencies were observed. Prevalence of low CSF HVA levels was significantly higher in neonatal patients (χ(2) =84.8, p<0.001). Abnormalities in white matter were associated with low CSF HVA (odds ratio 2.3, 95% confidence interval 1.5-3.5). INTERPRETATION: HVA abnormalities are observed in various neurological diseases, but some are probably an unspecific finding. No clear limits for CSF HVA values pointing towards primary diseases can be stated. We report several neurological diseases showing HVA alterations. No neuroimaging traits were associated with low HVA values, except for white matter abnormalities.

Increased concentrations of both NMDA receptor co-agonists D-serine and glycine in global ischemia: a potential novel treatment target for perinatal asphyxia.

Worldwide, perinatal asphyxia is an important cause of morbidity and mortality among term-born children. Overactivation of the N-methyl-D-aspartate receptor (NMDAr) plays a central role in the pathogenesis of cerebral hypoxia-ischemia, but the role of both endogenous NMDAr co-agonists D-serine and glycine remains largely elusive. We investigated D-serine and glycine concentration changes in rat glioma cells, subjected to oxygen and glucose deprivation (OGD) and CSF from piglets exposed to hypoxia-ischemia by occlusion of both carotid arteries and hypoxia. We illustrated these findings with analyses of cerebrospinal fluid (CSF) from human newborns affected by perinatal asphyxia. Extracellular concentrations of glycine and D-serine were markedly increased in rat glioma cells exposed to OGD, presumably through increased synthesis from L-serine. Upon reperfusion glycine concentrations normalized and D-serine concentrations were significantly lowered. The in vivo studies corroborated the finding of initially elevated and then normalizing concentrations of glycine and decreased D-serine concentrations upon reperfusion These significant increases of both endogenous NMDAr co-agonists in combination with elevated glutamate concentrations, as induced by global cerebral ischemia, are bound to lead to massive NMDAr activation, excitotoxicity and neuronal damage. Influencing these NMDAr co-agonist concentrations provides an interesting treatment target for this common, devastating and currently poorly treatable condition.

Effect of high-dose phenobarbital on oxidative stress in perinatal asphyxia: an open label randomized controlled trial.

OBJECTIVE: To evaluate the effect of high dose phenobarbital on lipid peroxidation and antioxidant enzymes in perinatal asphyxia. DESIGN: Open label, Randomized controlled trial. SETTING: Neonatal intensive care unit of a tertiary care teaching hospital. PARTICIPANTS: 72 full term inborn neonates with severe birth asphyxia. METHODS: Neonates were randomized to Study (phenobarbital) group and Control group. The infants in the study group received phenobarbital infusion (40 mg/kg) within first two hours of life while babies in the control group did not receive any phenobarbital. Rest of the management in both the groups was as per the unit protocol for the management of hypoxic ischemic encephalopathy. A cerebrospinal fluid examination was done at 12 ± 2 hours of life to determine the levels of superoxide dismutase, glutathione peroxidise and malonyldialdehyde. 60 neonates were followed up at 1 month of age when a detailed neurological examination was done. RESULTS: Four neonates in the study group and six neonates in the control group died during the study. Two neonates in the study group were lost to follow up. The cerebrospinal fluid lipid peroxides and antioxidant enzymes were significantly lower in the phenobarbital group as compared to the control group. The neurological outcome at one month follow up was found to be comparable between the two groups. CONCLUSION: Phenobarbital (40 mg/kg) given in the first two hours of life in term neonates with perinatal asphyxia led to a decrease in CSF levels of lipid peroxides and antioxidant enzymes at 12 ± 2 hours of life.

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.

Oxidative stress in perinatal asphyxia.

Oxygen free radicals may cause tissue injury in perinatal asphyxia. We measured plasma and cerebrospinal fluid levels of malondialdehyde and plasma levels of glutathione peroxidase, catalase, and superoxide dismutase in 50 term newborns with perinatal asphyxia and eight newborns without asphyxia. Neonates with sepsis, major congenital malformations, and hemolytic disease were excluded. The levels of plasma and cerebrospinal fluid malondialdehyde, as well as of plasma glutathione peroxidase, catalase, and superoxide dismutase, were significantly higher in newborns with perinatal asphyxia, and demonstrated a progressive increase with greater severity of hypoxic ischemic encephalopathy. Higher levels of plasma and cerebrospinal fluid malondialdehyde and plasma catalase were documented in newborns who died from hypoxic ischemic encephalopathy, compared with those who survived, but no such difference was found in plasma levels of glutathione peroxidase and superoxide dismutase. The data of the present study suggest that, despite the increased activities of antioxidant enzymes in perinatal asphyxia, these neonates experience higher degrees of oxidative stress, as evidenced by increased levels of plasma and cerebrospinal fluid malondialdehyde. Hence, oxygen free radicals can be considered to play a significant role in the pathophysiology of perinatal asphyxia.

The sex differences of cerebrospinal fluid levels of interleukin 8 and antioxidants in asphyxiated newborns.

Newborn males are more sensitive to brain injury than newborn females are. The aim of the present study was to find an explanation for this. We used the neuron-specific enolase (NSE) levels in the cerebrospinal fluid (CSF) for the classification of 32 newborns (19 males and 13 females) on their fifth postnatal day. The NSE levels were higher than normal (8.4 +/- 1.6 ng/mL) in 10 newborn males and 6 females and were, respectively, considered asphyxiated male and female groups. The remaining newborns, 9 males and 7 females, had normal CSF levels of NSE and were considered normal newborn male and female groups. The CSF samples were measured for 12 cytokines, using a cytokine array kit, and for total hydroperoxide and biological antioxidant potentials (BAPs), using the free radical analytic system. Among the 12 cytokines measured, only interleukin 8 (IL-8) was properly detected. The CSF levels of IL-8 were higher in the asphyxiated newborn females than in the other three groups. The mean CSF levels of BAPs in the asphyxiated newborn females were higher compared with the other three groups, but significance was detected only in comparison with the BAP levels in the CSF samples of the normal newborn males. There were no differences in total hydroperoxide levels among the groups. There are sex-related differences in the CSF levels of IL-8 and antioxidants in asphyxiated newborns, with higher levels in newborn females; this might contribute in the sexual dimorphism regarding the fact that females have better protection from brain injury than the males.

Excitatory amino acids and magnesium sulfate in neonatal asphyxia.

OBJECTIVE: The excitatory amino acids (EAA); glutamate and aspartate are released into the cerebrospinal fluids (CSF) of asphyxiated newborns. The objectives of this study were: (a) to examine the relation of the concentration of EAA in the CSF with the degree of brain injury, (b) To determine the time of the release of these EAA into the CSF, and (c) to detect the effect of magnesium sulfate (MgSO(4)) on their levels. DESIGNS AND METHODS. A randomized controlled trial was conducted on 47 full term asphyxiated newborns. Twenty three infants received an intravenous 10% solution of MgSO(4) at a dose of 250 mg/kg within the first 24h of life while the other 24 newborns received isotonic saline (0.9%) of an equal volume. Levels of glutamate and aspartate were measured before and 72 h after giving the trial solution. Results. In the study population (n=47) both glutamate and aspartate were significantly elevated in infants with higher grades of HIE compared to those with lower grades (P=0.013 and 0.031, respectively). Compared to baseline level, glutamate decreased significantly over time in placebo group (-8.28+/-14.26, P=0.025) and in MgSO(4) group (-14.39+/-18.72, P=0.005). Glutamate concentration did not differ between groups when measured at baseline (29.26+/-16.31 vs. 31.27+/-22.62, P=0.82) and at 72 h (19.28+/-15.63 vs. 19.6+/-16.54, P=0.87). The change in aspartate concentration over time was not significant in placebo group (-0.45+/-1.96, P=0.34) or in MgSO(4) group (-0.7+/-3.19, P=0.37). Aspartate did not differ between groups when measured at baseline (3.52+/-2.4 vs. 3.92+/-2.59, P=0.49) or at 72 h (2.79+/-1.24 vs. 3.05+/-2.48, P=0.92). Conclusions. The EAA; glutamate and aspartate are released in the CSF of asphyxiated newborns immediately after birth and declined by 72 h. Their initial concentrations correlated with the severity of HIE. Postnatal administration of MgSO(4) did not alter the levels of these 2 EAA.

[Levels of interleukin-6 and tumor necrosis factor-alpha in the cerebrospinal fluid of full-term newborns with hypoxic-ischemic encephalopathy]. / Níveis de interleucina-6 e fator de necrose tumoral-alfa no liquor de recém-nascidos a termo com encefalopatia hipóxico-isquêmica.

OBJECTIVE: To determine cerebrospinal fluid levels of interleukin-6 and tumor necrosis factor-alpha in full-term infants with hypoxic-ischemic encephalopathy, comparing with control infants. METHODS: Controlled, prospective study, performed between July 1999 and October 2001 with two groups of full-term newborns: 20 controls with no sepsis and/or meningitis and Apgar score > or =9 at first and fifth minutes; and cases, 15 asphyxiated full-term newborns with Apgar < or =4 and < or =6 at first and fifth minutes, umbilical blood cord pH<7.20 and/or umbilical arterial blood lactate>3.0 mmol/L, and requiring positive pressure ventilation for at least 2 minutes after birth. Cerebrospinal fluid samples were collected within 48 hours of birth for determination of interleukin-6 and tumor necrosis factor-alpha by enzyme immunoassay. RESULTS: Groups were similar concerning birthweight, gestational age, type of delivery and mean time required for cerebrospinal fluid sample collection. The samples were collected at mean with 17 hours of life. The medians cerebrospinal fluid levels in asphyxiated newborn infants were: 157.5 pg/ml for interleukin-6 and 14.7 pg/ml for tumor necrosis factor-alpha, significantly higher than the controls (interleukin-6: 4.1 pg/ml and tumor necrosis factor-alpha: 0.16 pg/ml). CONCLUSIONS: Full-term newborns with hypoxic-ischemic encephalopathy present higher cerebrospinal fluid interleukin-6 and tumor necrosis factor-alpha levels than the controls, possibly because of the local cerebral production of these cytokines, especially tumor necrosis factor-alpha. These results support a recommendation for future studies with brain blockers of the actions of these cytokines for neuroprotective strategies.

Clinical implication of the changes of cAMP, TXA2 and PGI2 in CSF of asphyxiated newborns.

To evaluate the changes of 3', 5'-cyclic adenosine monophosphate (cAMP), thromboxane A2(TXA2) and prostacyclin (PGI2) in cerebrospinal fluid (CSF) in the asphyxiated newborn and explore their roles in hypoxic-ischemic brain damage (HIBD). Thirty-six full term newborns were divided into 3 groups, including 12 with moderate-severe hypoxic-ischaemic encephalopathy (HIE), 13 with mild HIE, 11 without HIE (control group). The levels of cAMP, TXB2 (TXA2 metabolite) and 6-keto-PGF1 alpha (PGI2 metabolite) in CSF and plasma were measured 36-72 h after birth by RIA, and the concentrations were expressed as nM/L (cAMP), ng/L(TXB2 and 6-keto-PGF1 alpha). The infants were followed-up at 6 and 12 month of age and Mental Development Index (MDI) and Psychomotor Development Index (PDI) were measured using Bayley Scales of Infant Development (BSID). The CSF cAMP level in moderate-severe HIE group was 8.60 +/- 2.40, significantly lower than that of the mild HIE group (14.83 +/- 2.84) and the control group (24.43 +/- 2.39) (for both P < 0.01). The levels of TXB2 and 6-keto-PGF1 alpha in CFS in the moderate-severe HIE group (206.06 +/- 29.74, 168.47 +/- 23.02, respectively) were significantly higher than in the mild HIE group (83.37 +/- 28.57, 131.42 +/- 16.57, respectively, P < 0.01) and the control group (41.77 +/- 21.58, 86.23 +/- 13.05, respectively, P < 0.01). The level changes of cAMP, TXB2 and 6-keto-PGF1 alpha in plasma in all groups were similar to those in CSF, but no significant difference was found between mild HIE group and the control group (P > 0.05). The follow-up results showed that MDI and PDI of the moderate-severe HIE group were the lowest (84.79 +/- 13.34, 83.50 +/- 13.28, respectively), followed by mild HIE group (102.19 +/- 7.02, 99.94 +/- 9.08, respectively), with the control group being the highest (116.63 +/- 12.08, 116.69 +/- 10.87, respectively). Univariate analysis showed some significant difference (the moderate-severe HIE group vs. the mild HIE group or the control group, P < 0.01; the mild HIE group vs. the control group P < 0.05). The results suggested that the concentration of cAMP, TXA2 and T/K ratio in CSF after neonatal asphyxia might be sensitive markers in evaluating the severity of brain damage in early stage and predicting the future outcome.

Selective head cooling with hypothermia suppresses the generation of platelet-activating factor in cerebrospinal fluid of newborn infants with perinatal asphyxia.

Hypoxic-ischemic encephalopathy (HIE) remains one of the most important neurologic complications in the newborn. Several experimental and clinical studies have shown that hypothermia is the most effective means known for protecting the brain against hypoxic-ischemic brain damage. Furthermore, recent data have suggested that platelet-activating factor (PAF) could play a pathophysiologically important role in the progression of hypoxic-ischemic brain injury. The aim of the present study was to investigate the role of head cooling combined with minimal hypothermia in short-term outcome of infants with perinatal asphyxia. In addition, we have examined the effect of head cooling combined with minimal hypothermia on PAF concentrations in cerebrospinal fluid (CSF) after hypoxic-ischemic brain injury. The group of asphyxiated infants (Group 1) consisted of 21 full-term (gestational age >37 weeks). These infants were randomized and divided into either a standard therapy group (Group 1a; n=10) or cooling group (Group 1b; n=11). Head cooling combined with minimal hypothermia (rectal temperature 36.5-36 degrees C) was started as soon as practicable after birth. The infants were cooled for 72h and then were rewarmed at 0.5 degrees C/h. The control group (Group 2) consisted of seven full-term infants and none of these infants showed any sign of asphyxia. To measure PAF concentration in CSF, CSF with lumbar puncture was collected into tubes immediately before the cooling (1-3h after birth) and again after 36h. We had no evidence of severe adverse events related to hypothermia. In Group 1a, two infants died after 72h of life; however, all newborn infants in Group 1b survived. Convulsion required treatment in three infants of standard therapy group (1a); none of the infants in Group 1b had clinical seizure activity. Abnormal EEG patterns were found in four infants of Group 1a; no EEG abnormalities were noted in Group 1b (P<0.05). On admission (before cooling), PAF concentration in CSF of asphyxiated infants was found to be significantly higher when compared with that of control (P<0.001). Mean PAF concentration before initiation of the study was similar in the two asphyxiated groups (Group 1a vs. 1b) (P>0.05). Obtained PAF level in CSF after 36h, showed a profound decline in cooling group of infants compared to Group 1a infants (P<0.01). In conclusion, the present study suggests that cerebral cooling with minimal hypothermia started soon after birth has no severe adverse effects during 72-h cooling period and that short-term outcome of infants are encouraging. Our results also support the hypothesis PAF an important mediator in hypoxic-ischemic brain injury and demonstrate that head cooling combined with minimal hypothermia reduces the normal increase in PAF following hypoxic-ischemic brain injury in full-term infants.