Levels of interleukin-6 and tumor necrosis factor-alpha in the cerebrospinal fluid of full-term newborns with hypoxic-ischemic encephalopathy

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1 /03/79-04/297 Jornal de Pediatria Copyright 2003 by Sociedade Brasileira de Pediatria ORIGINAL ARTICLE Levels of interleukin-6 and tumor necrosis factor-alpha in the cerebrospinal fluid of full-term newborns with hypoxic-ischemic encephalopathy Rita de Cássia Silveira, 1 Renato S. Procianoy 2 Abstract 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 > 9 at first and fifth minutes; and cases, 15 asphyxiated full-term newborns with Apgar < 4 and < 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: 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. J Pediatr (Rio J) 2003;79(4): : hypoxic-ischemic encephalopathy, perinatal asphyxia, interleukin-6, tumor necrosis factor-alpha, cytokines. 1. PhD. Neonatologist, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul. 2. Professor, Universidade Federal do Rio Grande do Sul. Head of the Neonatology Service, Hospital de Clínicas de Porto Alegre. Financially supported by: CNPQ and FIPE-HCPA (Fund of Research Support of Hospital de Clínicas de Porto Alegre). Manuscript received Feb , accepted for publication Mar Introduction Despite recent advances in biophysical and biochemical monitoring of the fetus during labor and delivery, perinatal asphyxia still contributes heavily to neonatal morbidity and mortality, affecting 2 to 5 newborns for every 1,000 live births. 1,2 It is the result of a number of conditions which can 297

2 298 Jornal de Pediatria - Vol. 79, Nº4, 2003 Levels of interleukin-6 and tumor necrosis factor-alpha... Silveira RC et alii interfere with fetus-placenta gaseous exchange leading to progressive hypoxemia, hypercapnia and metabolic acidosis. The clinical result is a hypoxic-ischemic lesion to the brain of the neonate which develops Hypoxic-Ischemic Encephalopathy (HIE), the most often described and best known clinical manifestation of perinatal asphyxia. 3,4 The pathophysiological mechanisms of HIE have become the subject of recent interest, with the principal aim of developing neuroprotective strategies based on biochemical, cellular and humoral knowledge of cerebral lesions. There is a great deal of diverse evidence indicating that the inflammatory cascade is involved in the pathogenesis of ischemic cerebral lesions. The inflammatory reaction produced by ischemia in the central nervous system (CNS) is characterized by an influx of leukocytes, including polymorphonuclear leukocytes, followed by monocytes, and by the activation of the microglia, for which the expression of specific adhesion molecules, chemotactile factors and proinflammatory cytokines is necessary. Cytokines are proteins with a low molecular weight, produced and secreted by monocytes, macrophages, endothelial cells and fibroblasts and in the CNS they are produced and secreted by the microglia and astrocytes. 5-7 Experimental models have suggested that a number of different cytokines, primarily interleukin-6 (IL-6) and tumor necrosis factor- a (TNF-α), are involved in ischemic cerebral damage Interleukin-1 ß (IL-1 ß) and O TNF-α are observed at elevated levels after cerebral ischemia and can induce an inflammatory reaction of the CNS, in conjunction with IL-6. Furthermore, they appear to directly modulate the CNS cells apoptosis process, promoting differentiation, proliferation and subsequent infiltration by leukocytes A direct correlation between cerebrospinal fluid IL-6 and TNF-α levels and neurological prognosis after acute cerebral ischemia has been demonstrated in adults. 15 In full-term newborns with hypoxic-ischemic encephalopathy (HIE), the levels of TNF-α and IL-1 ß in the cerebrospinal fluid were greater in patients who presented neurological alterations at 12 months. 16 Martín-Ancel et al., found that cerebrospinal fluid IL-6 levels were significantly higher in newborns with severe neurological manifestations than in those with mild or moderate encephalopathy, however, these authors did not assess the concentration of TNF-α in cerebrospinal fluid. 17 The majority of studies which have investigated the mechanisms involved in cerebral lesions after perinatal asphyxia are experimental and clinical studies of newborns with HIE which investigate the behavior of IL-6 and TNF-α - in the CNS are necessary since it is believed that these two cytokines are more identified with the inflammatory processes of neonatal hypoxia-ischemia. The intention of this study was to investigate whether IL-6 and TNF-α aconcentrations in the cerebrospinal fluid of full-term neonates with HIE are higher than in normal neonates (controls). Patients and Methods A case-control study was performed which included all full-term neonates born at the Obstetrics Unit of the Hospital de Clínicas de Porto Alegre and admitted to the neonatal ICU, with a diagnosis of perinatal asphyxia or requiring lumbar puncture during the first 48 hours of life to investigate early onset neonatal sepsis, during the period July 1999 to October A diagnosis of perinatal asphyxia was made in the presence of at least three of the following criteria, with criteria 3 and 4 being obligatory: 1,18,19 1.signs of fetal suffering revealed by intrapartum monitoring (IPM) such as; persistent decelerations, sustained fetal bradycardia or silent IPM ; 2. Apgar score less than or equal to 4 during the first minute and less than or equal to 6 at the fifth; 3.umbilical ph value less than 7.20; 4. Umbilical cord arterial lactate greater than 3.0 mmol/l; 5.need for positive pressure ventilation for at least two minutes in order to start respiration. The diagnosis of hypoxic-ischemic encephalopathy was established by the presence of perinatal asphyxia associated with neurological manifestations resulting from hypoxemia and ischemia. 16 HIE was classified in accordance with the criteria defined by Sarnat and Sarnat. 20 The control group was made up of non-asphyxiated neonates with Apgar scores > 9 at the first and fifth minutes of life, who had required a cerebrospinal fluid examination for investigation of neonatal sepsis, with antibiotic therapy being suspended in less than 48 hours and clinical evolution being favorable. This group had their blood tests and blood cultures performed by the treating doctor before antibiotic therapy began, presented normal results and their laboratory examinations were conducted due to initial suspicion of early neonatal sepsis. Newborns were excluded if there was a clinical or laboratory-based suspicion of congenital infection, if sepsis and/or meningitis were presented, if there were congenital malformations, if there were complications during the spinal tap for cerebrospinal fluid examination, if there were convulsive crises which were unrelated to hypoxic events and whose etiology was unclear, if the mother had a history of drug abuse or had any infection from the STORCH group during pregnancy or if she was positive for the human immunodeficiency virus (HIV+) or, finally, if opiates or respiratory depressive drugs had been administered during peripartum. Both the control-group neonates and the cases were observed throughout internment until discharge, and for the HIE cases the following were also performed: neuropediatric observation, electroencephalogram and transfontanelar cerebral ultrasound. An additional volume of cerebrospinal fluid was obtained from the sample requested by the maternity team as part of their routine investigations and as such no samples were taken exclusively for the purposes of research. Cerebrospinal

3 Levels of interleukin-6 and tumor necrosis factor-alpha... Silveira RC et alii Jornal de Pediatria - Vol. 79, Nº4, fluid was obtained by lumbar puncture (500 µl total volume) and frozen at - 70 C. The samples were taken during the first 48 hours of the neonates lives for later IL-6 and TNF-α assays. All samples were double-tested. The technique employed was IL-6 and TNF-α enzyme immunoassay (Quantikine Human IL-6 and TNF-α, R & D Systems, Inc. MN, USA). The limit set for IL-6 detection was 0.7 pg/ml and for TNF-α 0.1pg/ml, with IL-6 and TNF-α inter-assay and intra-assay coefficients of variation of less than 5%. Readings were taken with a Spectramax automatic optical at a wavelength of 570 nm. Statistical analysis: Sample size was calculated to give a power of 0.97 and a significance level of Results were expressed as mean and standard deviation or median and variation. The Chi-square test was used to compare categorical values, the Student t test for parametric data and the Mann-Whitney test for the comparison of cerebrospinal fluid cytokine levels across the two groups. The significance cut-off was set at p < The research protocol was approved by the Ethics Committee of our institution and in all cases informed consent was obtained from the parent or guardian. Results Thirty-five newborns were included (n = 35), of whom fifteen were cases and twenty were controls. There were no differences between the cases and the controls in terms of birth weight, gestational age, classification by weight and gestational age, type of delivery and average time at which cerebrospinal fluid was collected. There was no significant difference between the groups in terms of the time which had passed between birth and cerebrospinal fluid collection. Both groups had their samples taken at an average of 17 hours of life and the medians for hours of life were 8 (2-25) for the asphyxiated group with HIE and 10 (3-26) for the controls. Apgar scores were significantly lower among the asphyxiated newborns (Table 1). Table 1 - Characteristics of the population studied Group HIE Control N Birth weight (g) 3,439 ± 320 3,046 ± 118 Gestational age (weeks) 39.6 ± ± minute Apgar* 1 (0-4) 9 (9-10) 5-minute Apgar* 5 (0-6) 9 (9-10) Vaginal delivery 12 (80%) 14 (70%) AGA 11 (73%) 14 (70%) Collection moment (life period) 8 (2-25) 10 (3-26) Data shown in mean and standard deviation or median and variation. *p < AGA: adequate for gestational age. The collection of cerebrospinal fluid from the members of the control group was due to one of the following situations: untreated or incompletely treated maternal urinary infection in four cases; premature membrane rupture (more than 24 hours) in eight cases; five newborns had fever; and three had respiratory discomfort, diagnosed as transitory tachypnea during clinical work-up. The asphyxiated newborns were classified into three HIE stages, according to criteria developed by Sarnat and Sarnat as follows; first degree HIE was observed in eight neonates (53%), second degree HIE in three (20%) and third degree HIE in four newborns (27%). Cerebral ultrasound was performed for all of the asphyxiated patients, revealing alterations in five cases (33%) Diffuse cerebral edema and thalamic hyperechogenecity were the most common alterations. Of the 14 patients for whom electroencephalogram was performed, six presented significant alterations which were attributable to HIE (43%), and the neurological examination revealed alterations either before discharge or at some point before death in six cases. There were three deaths, all of neonates who had severe HIE. Data obtained exclusively from the newborns with HIE is summarized in Table 2. Levels of IL-6 in cerebrospinal fluid were significantly higher in the asphyxiated newborns when they were compared with control neonates. The differences in cerebrospinal fluid TNF-α levels were even more obvious. Concentrations were significantly greater in asphyxiated neonates and practically undetectable in the cerebrospinal fluid of the controls (Table 3). Discussion Cytokines have been implicated in a number of different mechanisms which could potentialize a an ischemic cerebral lesion, such as: liberation of the inducible form of the enzyme Nitric Oxide Synthase by astrocytes; the recruitment, activation and adhesion of leukocytes to the endothelium; the promotion of a pro-coagulatory endothelial state and the regulation of the apoptosis process. Interleukin-6 and TNFα are both cytokines which actively participate in the cerebral lesion mechanisms which occur during perinatal asphyxia. 16,17,21 The results of our study demonstrate these findings in human neonates with HIE and allow the concentrations in their cerebrospinal fluid to be compared with those of controls. Cytokines, particularly IL-6, affect the differentiation, growth and survival in vitro of neuronal cells, and as a consequence, the newborn brain is particularly susceptible to alterations to cytokine concentrations. 22 Our samples were obtained, on average, at 17 hours postpartum, a fact which is extremely relevant to the study because the half-life of cytokines in plasma is very short, they are produced and secreted in response to a variety of stimuli and have a very early peak in serum. The half-life of

4 300 Jornal de Pediatria - Vol. 79, Nº4, 2003 Levels of interleukin-6 and tumor necrosis factor-alpha... Silveira RC et alii Table 2 - Characteristics of asphyxiated newborns Patient U-sound * EEG Exam HIE Umbilical cord Umbilical cord Deaths ph lac (mmol/l) 1 N A N No 2 N N N No 3 N N No 4 A A A Yes 5 N N A No 6 A N N No 7 N N N No 8 N N N No 9 A A A Yes 10 N N N No 11 N A A No 12 N N N No 13 N N N 1 6, No 14 A A A No 15 A A A Yes * Ultrasound; Electroencephalogram; Neurological examination at discharge/death; Stages of hypoxic-ischemic encephalopathy; A altered; N normal. cytokines in cerebrospinal fluid is little known, but it is believed that they behave similarly. 5,6 In adults who had suffered ischemic vascular accidents, IL-6 levels were found to be higher in the cerebrospinal fluid than in the blood during the first 2 to 3 days after the insult. Over time these levels achieve equilibrium, suggesting early intrathecal IL-6 production. 23 The half-life of IL-6 in the asphyxiated neonate is much shorter than in an adult with an ischemic cerebral lesion, probably as a result of differences in the level of maturity of the immune system. 17 Tumor Necrosis Factor-a, however, begins to increase during the first hour after occlusion of the middle cerebral artery, with response to ischemia peaking in between 6 and 12 hours. 24,25 For this reason our concern to collect cerebrospinal fluid samples as quickly as possible was fundamental to an adequate analysis of the results. Table 3 - Cerebrospinal fluid IL-6 and TNF-α levels Group EHI Control p N IL-6 (pg/ml) ( ) 4.1 ( ) < TNF-α (pg/ml) 14.7 ( ) 0.16 (0-0.25) < Data shown in median and variation. Mann-Whitney test. Our results revealed elevated IL-6 levels in the cerebrospinal fluid of newborns who had suffered perinatal asphyxia when compared with controls. We took care to exclude from the control group newborns who had been subjected to any kind of pathological situation which could have caused an inflammatory reaction and resulted in elevated CNS cytokine levels. The exact origin of IL-6 during perinatal asphyxia still remains to be ascertained. It is also unclear in what way IL-6 participates in the degeneration or repair of neurons after perinatal asphyxia. Astrocytes and microglia are possible sources of CNS IL Monocytes and neutrophils, recruited during cerebral ischemia, are capable of producing IL-6 in response to stimuli from TNF-α and IL-1ß. 5,27 This would also explain the highly significant elevation of cerebrospinal fluid TNFα levels in the asphyxiated newborns in our study. Newborns who have suffered transitory hypoxiaischemia during an episode of asphyxia while being born, can appear relatively normal soon after resuscitation in the delivery room, presenting evidence of the cerebral lesion some hours later, as is the case with the convulsions which characteristically occur during the first 24 hours of life. 28,29 This retarded cerebral lesion mechanism is not yet completely clear, but apoptotic cells have been found during cerebral necropsies of neonates who had died of perinatal asphyxia. It is probable that the pathophysiology of perinatal asphyxia is a two-phase process which is closely related to apoptosis mechanisms. 30 Apoptosis could possibly be induced by

5 Levels of interleukin-6 and tumor necrosis factor-alpha... Silveira RC et alii Jornal de Pediatria - Vol. 79, Nº4, TNF-α as a result of the activation of sphingomyelinase, resulting in increased cytosolic ceramide concentrations, which potentializes apoptosis. 30 Oygür et al. evaluated the predictive power of cerebrospinal fluid TNF-α and IL-1ß levels for prognosis of full term newborns with HIE and they are both good predictors of early sequelae. The concentrations of TNF-α in the cerebrospinal fluid of neonates who died were much greater soon after the hypoxic insult, reinforcing the theory that TNF-α is responsible for the two-phase process of the cerebral lesion and unfavorable prognosis due to its induction of apoptosis in neuronal cells. 16 The association between severity and frequency of deaths and cerebrospinal fluid IL-6 and TNF-α levels was not investigated because only three of the newborns died which did not allow statistical analysis. Notwithstanding, all cases which ended in death presented third degree HIE and more elevated levels of these markers, demonstrating a tendency which it would be possible to prove with a greater number of cases. The sample size calculation was made to analyze the differences in cerebrospinal fluid IL-6 and TNF-α levels between patients and controls and not to establish this association. Interleukin-6 and TNF-α appear to play an important role in the cascade of inflammatory events in perinatal asphyxia. Full term newborns with HIE presented higher levels of IL-6 and TNF-α in their cerebrospinal fluid, attributable to a direct action of the asphyxiating stimulus on the central nervous system. The local, cerebral production of TNF-α and IL-6 is probable in newborns who have suffered a hypoxic-ischemic insult and makes possible future studies aimed at developing new therapeutic methods such as cerebral blockers of the action of these cytokines. We believe, nonetheless, that this study will contribute to the development of new strategies for the neuroprotection of neonates with hypoxic-ischemic encephalopathy. Acknowledgements The authors would like to thank the teaching doctors at UFRGS: Célia Carlini and Mário Wagner and the practicing neonatologist, Clarissa Miura. References 1. Chou YH, Tsou Yau KI, Wang PJ. Clinical application of the measurement of cord plasma lactate and pyruvate in the assessment of high-risk neonates. Acta Paediatr 1998;87: Mulligan JC, Painter MJ, O Donoghue PA, MacDonald HM, Allan AC, Taylor PM. Neonatal asphyxia. II. Neonatal mortality and long-term sequelae. J Pediatr 1980;96: Lupton BA, Hill A, Roland EH, Whitfield MF, Flodmark O. Brain swelling in the asphyxiated term newborn: pathogenesis and outcome. Pediatrics 1988;82: Williams CE, Mallard C, Tan W, Gluckman PD. Pathophysiology of perinatal asphyxia. Clin Perinatol 1993;20(2): Clark WM. Cytokines and reperfusion injury. 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6 302 Jornal de Pediatria - Vol. 79, Nº4, 2003 Levels of interleukin-6 and tumor necrosis factor-alpha... Silveira RC et alii 26. Maeda Y, Matsumoto M, Hori O, Kuwabara K, Ogawa S, Yan SD, et al. Hypoxia/reoxygenation-mediated induction of astrocyte interleukin 6: a paracrine mechanism potentially enhancing neuron survival. J Exp Med 1994;180: Silverstein FS, Barks JD, Hagan P, Liu XH, Ivacko J, Szaflarski J. Cytokines and perinatal brain injury. Neurochem Int 1997;30: Rivkin MJ, Volpe JJ. Hypoxic-ischemic brain injury in the newborn. Semin Neurol 1993;13: Ahn OM, Korst LM, Phelan JP, Martin GI. Does the onset of neonatal seizures correlate with the timing of fetal neurologic injury? Clin Pediatr (Phila) 1998;37: Mehmet H, Edwards AD. Hypoxia, ischaemia, and apoptosis. Arch Dis Child Fetal Neonatal Ed 1996;75:F73-5. Corresponding author: Rita de Cássia Silveira Rua General João Telles, 542/601 CEP Porto Alegre, RS, Brazil rita.c.s@terra.com.br