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Hypoxic-ischemic brain injury after severe birth asphyxia remains a clinical problem in spite of improvements in obstetric and neonatal care(1). The pathophysiology is complex, and even if a number of critical factors have been defined, the mechanisms of brain injury are largely unknown(2–4). It is of crucial importance to increase the understanding of the events leading to brain injury and to identify early markers predicting outcome after birth asphyxia.

Experimental studies indicate that the immunoinflammatory system is involved in the biochemical cascade leading to injury in the immature brain after hypoxia-ischemia(5, 6). Neutrophils accumulate within the vascular wall, and microglia are activated during early reperfusion(7, 8). In addition, the expression of mRNA and bioactive protein for the proinflammatory cytokines IL-1, IL-6, and TNF-α increase 1-4 h after hypoxia-ischemia(9, 10). Furthermore, reduction of neutrophil accumulation by administration of anti-neutrophil serum(8) or inhibition of the effect of IL-1 with IL-1 receptor antagonist attenuate perinatal brain injury(10, 11).

Studies on human stroke cases have shown that the CSF levels of IL-1β and IL-6 increase considerably after the insult, exceeding the content in serum 2 d after onset of symptoms(12). Recently, a marked increase of IL-10, IL-8, and GM-CSF was found to occur after stroke(13), indicating that both pro-and antiinflammatory cytokines are produced in response to brain ischemia. The properties of the immune system(14, 15) and the inflammatory response to insults(16) change during maturation, which makes it important to specifically evaluate the role of cytokines for development of perinatal brain injuries(17). To our knowledge, there are no published reports on the possible role of cytokines in human newborns suffering from asphyxia.

The purpose of the present study was to evaluate the CSF contents of proinflammatory (IL-1, IL-6, IL-8, TNF-α, GM-CSF) and immunoregulatory(IL-10) cytokines after birth asphyxia and relate the levels to the degree of HIE and short-term outcome. The choice of cytokines was based on data available from experimental studies as well as studies of cases of human stroke.

METHODS

Patients. Twenty-seven infants with gestational age ≥35 wk treated at the neonatal intensive care unit at Karolinska Hospital from November 1990 to August 1996 were included in the study. Twenty infants fulfilled the following criteria for birth asphyxia: 1) intrapartum distress as indicated by the cardiotocographic pattern (late decelerations for>1 h or severe abnormalities such as absent variability or bradycardia for>30 min before birth), early passage of thick meconium or scalp pH < 7.2 immediately before birth, and 2) need for neonatal resuscitation with positive-pressure ventilation for >3 min, 5-min Apgar score ≤6, or umbilical arterial/first postnatal pH < 7.1 and/or base excess ≤ -10.

Infants with CNS malformations, macroscopic blood in the CSF, or systemic infections with positive bacterial or viral cultures from blood and/or CSF were excluded. The asphyxiated group was further divided into mild, moderate, and severe HIE according to Sarnat and Sarnat(18). Infants with moderate or severe HIE were examined by computerized tomography scan of the brain after 3 d and by early diagnostic EEG and/or continuous amplitude-integrated EEG (CFM, Lectromed®).

The control group consisted of seven infants with clinical signs of infection but with negative bacterial and viral cultures from blood and CSF and no findings suggesting CNS pathology. The asphyxiated infants were examined by a neuropediatrician at 6, 10, 18, and 48 mo of age. The infants in the control group were followed as indicated at discharge, and information on these children was also obtained from well baby clinics and from the parents. Disabling abnormalities in tone or reflexes, seizures, or blindness were classified as abnormal outcome.

CSF sampling. CSF was obtained by spinal taps within 72 h of delivery. After 1.5 mL of CSF for clinical use had been tapped, the next 1.5 mL of CSF were collected and spun at 4000 rpm at 4 °C for 10 min. The supernatant was then portioned into three tubes, and the samples were stored in -70 °C until analyzed. IL-6, IL-8, GM-CSF, IL-1β, and IL-10 were analyzed in 7 control and 20 asphyxiated infants, whereas TNF-α was determined in 7 control and 17 asphyxiated infants. Parental consent was obtained in each case for the extra amount of CSF taken, and the study was approved by the local ethics committee of the hospital.

Cytokine analysis. IL-6 was analyzed using a bioassay. Cell line B 13.29, which is dependent on IL-6 for growth, has been previously described(19). For the IL-6 determinations, the more sensitive subclone B9 was used(20, 21). B9 cells were harvested from tissue culture flasks, seeded into microtiter plates(Nunc, Roskilde, Danmark) at a concentration of 5000 cells/well, and cultured in Iscove's medium supplemented with 5 × 10-5 M 2-mercaptoethanol, 5% FCS (Seralab, Sussex, UK), penicillin (100 U/mL), and streptomycin (100 mg/mL), and CSF samples were added. [3H]Thymidine was added after 68 h of culturing, and the cells were harvested 4 h later. The samples were tested in 2-fold dilutions and compared with a recombinant human IL-6 standard (Genzyme, Cambridge, MA). B9 cells were previously shown not to react with several recombinant cytokines, including IL-1α, IL-1β, IL-2, IL-3, IL-5, GM-CSF, TNF-α, and interferon-γ. There was only weak reactivity with IL-4(21). To assess the specificity of the bioassay we have used a highly purified MAb specific for human IL-6(Genzyme, Cambridge, MA) in an neutralization assay. Preincubation of 10 mg/mL of this antibody with either recombinant IL-6 or CSF from patients containing IL-6 (1 h, 37 °C) reduced proliferative responses of B9 indicator cells by on an average ≥95%.

Levels of IL-1β in CSF samples were estimated by an ELISA (Quantikine R&D Systems, Minneapolis, MN). Levels of GM-CSF in CSF samples were quantified by an ELISA using GM-CSF-specific unlabeled MAbs (Pharmingen, San Diego, CA) for coating (2 mg/mL) of polystyrene flat-bottom plates (4 °C, overnight), followed by a wash with Tris, 0.05% Tween 20 and blocking with 1% BSA in Tris-NaCl for 1 h at 37 °C. After emptying, the plates were incubated for 2 h at 37 °C with CSF samples diluted 1:5 in Tris-NaCl containing 1% BSA. Biotinylated GM-CSF-specific MAb (Pharmingen) were added and incubated overnight at 4 °C. The plates were washed again and incubated with ExtrAvidin alkaline phosphatase (Sigma Chemical Co., St. Louis, MO). The enzymesubstrate was then added, and OD was determined in a Titertek Multiscan Photometer (Flow Laboratories, McLean, VA) at 405 nm. The concentration of GM-CSF was calculated using a standard curve based on known quantities of human recombinant GM-CSF (Genzyme, Cambridge, MA).

Levels of IL-10 were quantified in basically the same way as for GM-CSF. Monoclonal unlabeled (4 mg/mL) and biotinylated (4 mg/mL) antibodies specific for IL-10 were purchased from Pharmingen, whereas a recombinant human IL-10 standard was obtained from R&D Systems (Minneapolis, MN).

Levels of IL-8 were quantified using 10 mg/mL goat anti-human IL-8 (R&D Systems) in bicarbonate buffer as a solid phase coating. The developing antibodies were rabbit anti-human IL-8 (Endogen, Cambridge, MA) followed by alkaline phosphatase-labeled goat anti-rabbit IgG (Sigma Chemical Co.) diluted 1:1000 in PBS-Tween 20 containing 1% BSA. Human recombinant IL-8 (R&D Systems) was used to create the standard curve.

Levels of TNF-α were quantified using 2.5 μg/mL monoclonal mouse anti-human TNF-α (Endogen, Cambridge, MA) in bicarbonate buffer, as a solid phase coating. The developing antibodies were biotin-labeled monoclonal mouse anti-human TNF-α (Endogen) followed by streptavidin alkaline phosphatase diluted 1:1000 in Tris-Tween 20 and enzyme substrate. Human recombinant TNF-α (Endogen) was used to create the standard curve.

For the different cytokines the lowest detectable levels were as follows: IL-6, 25 pg/mL; IL-8, 10 pg/mL; IL-10, 15 pg/mL; TNF-α, 0.9 pg/mL; GM-CSF, 10 pg/mL; and IL-1β, 1 pg/mL. All cytokine values below the detection limit were set to zero.

Statistical analysis. All results are given as medians and interquartile ranges unless stated otherwise. The Mann-Whitney U test was used to test differences between the asphyxia group and the control group. The Kruskal-Wallis test was used to test the association between cytokine level and the degree of HIE or clinical outcome. The Spearman rank test was used for evaluation of correlations.

RESULTS

Patients. Group data on the asphyxia group and the control group are given in Table 1. The infants in the asphyxia group (n = 20) were classified as having severe (n = 8), moderate (n = 8), and mild (n = 4) HIE. Four infants in the group with severe HIE died in the neonatal period. The remaining four infants have been followed for 7-24 mo, and all have an abnormal outcome with athetoid and tetraplegic cerebral paresis and/or severe seizure disorders. The eight infants with moderate HIE have been followed 6-48 mo. Two infants have a definite abnormal outcome with diplegia and tetraplegia, respectively. Two infants have a slight delay in motor function at 16 mo of age, but do not fulfill the criteria for abnormal outcome. In the group with mild HIE, followed 12-17 mo, one infant has developed hemiplegia and a follow-up computerized tomography scan of the brain demonstrated a cerebral infarction of probable perinatal origin, whereas the others were normal. The infants in the control group have been followed 5-48 mo and are all normal.

Table 1 Clinical data on the studied infants
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CSF cytokine levels. The concentrations of IL-6 and IL-8 were considerably higher in the asphyxia group compared with the control group. For IL-6 the level was 250 (35-542.5) pg/mL compared with 0 (0-18.8) pg/mL(p = 0.001), and for IL-8 the levels were 170 (70-1440) pg/mL compared with 10 (0-120) pg/mL (p = 0.009). There were no significant differences found between asphyxiated infants and control subjects for any of the other cytokines analyzed (data not shown). TNF-α was detectable in 3/7 control and in 8/17 asphyxiated infants. The median concentration of TNF-α was 1.5 pg/mL in the control group and 1.5 pg/mL in the asphyxia group.

The levels of the cytokines in infants with varying degrees of HIE are demonstrated in Table 2. There was a significant correspondence between the levels of IL-6 and IL-8 and the degree of encephalopathy. The distribution of the individual values are shown in Figures 1 and 2.

Table 2 Cytokine levels in CSF for control group and different HIE groups
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Figure 1
figure 1

IL-6 levels in CSF for control subjects and infants with HIE. CSF concentrations of IL-6 in the control (n = 7) and the asphyxiated (n = 20) group. Individual IL-6 values are plotted for the control group and the asphyxiated infants separated into mild HIE I(n = 4), moderate HIE II (n = 8), and severe HIE III(n = 8). Values are expressed as picograms/mL, and horizontal bars indicate medians. There was a significant association between the concentration of IL-6 and the degree of HIE (p = 0.03; Kruskal-Wallis test).

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Figure 2
figure 2

IL-8 levels in CSF for controls and infants with HIE. CSF concentrations of IL-8 in the control (n = 7) and the asphyxiated (n = 20) group. Individual IL-8 values are plotted for the control group and the asphyxiated infants separated into mild HIE I(n = 4), moderate HIE II (n = 8), and severe HIE III(n = 8). Values are expressed as picograms/mL and horizontal bars indicate medians. There was a significant association between the concentration of IL-8 and the degree of HIE (p = 0.02; Kruskal-Wallis test).

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The levels of different cytokines in asphyxiated infants with varying outcome are demonstrated in Table 3 and Fig. 3. There was a significant difference in IL-6 levels between groups with different outcome (Fig 3). The relation between IL-8 and outcome, demonstrated in the same figure, did not reach statistical significance. No significant associations were found between outcome and the levels of IL-10 or GM-CSF (Table 3).

Table 3 Cytokine levels for asphyxiated infants with different outcome
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Figure 3
figure 3

IL-6 and IL-8 levels in CSF for infants with different outcomes. The relationship between the CSF cytokine levels and outcome(normal, abnormal, and dead) for infants in the asphyxia group (n = 20). The IL-6 and IL-8 levels are expressed as picograms/mL, and values are given as medians ± IQR. The levels of IL-6 were significantly higher(p = 0.05) in the abnormal/dead groups compared with normal subjects(Kruskal-Wallis test). The IL-8 concentration was not significantly associated with outcome.

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Only four infants had detectable levels of IL-1β. Three of those had severe HIE, two died and one developed severe seizures. One infant with mild HIE and a detectable level of IL-1β developed hemiplegia secondary to a cerebral infarction of probable perinatal origin.

DISCUSSION

The main findings of the present study were that CSF levels of IL-6 and IL-8 increase considerably after birth asphyxia, and that these increases were more pronounced in the infants with severe clinical course and poor prognosis. IL-1β was increased in a small number of infants, all of whom had an abnormal outcome.

One of the questions to be discussed is whether the cytokines were produced by cells in the CNS or rather originated from the systemic compartment. Systemically produced cytokines could enter into the CSF through a damaged blood-brain barrier. The protein content was not significantly different in CSF from asphyxiated infants compared with control infants, but a difference in the degree of blood-brain barrier damage cannot be excluded, because the protein content was not analyzed in all cases. A previous study of stroke patients has also demonstrated that the cytokine levels in CSF did not differ between patients with blood-brain barrier damage and those with no damage(12). Birth asphyxia affects, however, the entire body and is often associated with multiple organ failure, which may hypothetically result in a substantial systemic increase of cytokines. Therefore, in the absence of simultaneous blood samples, we cannot at present exclude a systemic contribution, especially as some cytokines may penetrate even across an intact blood-brain barrier(22). An intrathecal production appears, however, to be the more likely explanation in light of the facts that cerebral hypoxia-ischemia induced in immature and adult animals is associated with a considerable expression of cytokines in the brain tissue at the mRNA and protein level(9, 10, 23) and that these cytokines are known to be produced by microglia, endothelial cells, and astrocytes under such conditions(24–26).

There was a significant difference in the timing of the CSF sampling between the asphyxiated (13.5 h after birth) and the control (36 h after birth) infants that may have affected the results. The time course of the cytokine response after birth asphyxia is not known, but the CSF levels of IL-6, IL-8, and IL-1β were all significantly elevated within 24 h after the start of symptoms in stroke patients and reached peak levels after 2-3 d(12, 13), suggesting that we rather may have underestimated the difference between asphyxiated and control subjects. Furthermore, the significant correspondence between cytokines and HIE or between cytokines and outcome cannot be attributed to a bias in timing of sampling.

Irrespective of the possible origins of these cytokines it is important to consider the possible effects of IL-6, IL-8, and IL-1β accumulated after birth asphyxia. We found presently a considerable increase of IL-6 in CSF of asphyxiated infants, and the concentration corresponded to the degree of HIE and to short-term outcome. This is in agreement with a previously reported increase of IL-6 in the serum and CSF of stroke patients(12, 27) and a significant correlation between IL-6(1-3 d after onset of stroke) and volume of brain lesions(12). IL-6 is generally considered as a proinflammatory cytokine, as the levels correlate to the severity of the infectious disease, the production of IL-6 is often triggered by IL-1, and these two cytokines respond to the same pathologic insults(28, 29). The IL-6 increase is, however, more protracted, and significant concentrations appear in the blood stream(25). IL-6 is a multifunctional cytokine(30) and in contrast to IL-1β, IL-6 lacks direct neurotoxicity(29), activates anti-proteases(29), inhibits lipopolysaccharide-induced TNF-α(31), and stimulates circulating IL-1 receptor antagonist and soluble TNF receptor(32), suggesting that IL-6 may also have antiinflammatory properties. Recently, IL-6 was found to suppress demyelination(33) and to reduce N-methyl-d-aspartic acid receptor toxicity and ischemic damage in vivo in adult animals(34). Due to the multifarious abilities of IL-6 it is not possible at present to define its role in the injury response to hypoxiaischemia.

We found also high concentrations of IL-8 in CSF of most asphyxiated infants, which agree with recently observed changes in CSF from stroke patients(13) and elevation of the rat IL-8 analog cytokine-induced neutrophil chemoattractant in brain tissue after ischemia in adult animals(23). IL-8 belongs to a group of chemokines that act predominantly on neutrophils as mediators of inflammation and are produced by the monocyte/macrophage lineage, activated T cells, and endothelial cells(35). In hypoxia-ischemia, IL-8 may be anticipated to play an essential part in neutrophil activation, i.e. invasion of these cells into the brain tissue and the initiation of degranulation and phagocytosis(36). Indeed, studies in newborn rats suggest that neutrophils accumulate after hypoxia-ischemia with an 8-fold increase of the myeloperoxidase activity during early reperfusion and anti-neutrophil serum attenuates brain injury(8), suggesting that an increase of IL-8 may be of pathophysiologic significance.

In the present study IL-1β was detectable in only four patients who all had an adverse outcome, three with severe HIE and one with cerebral infarction. IL-1β acts locally in an apocrine fashion, and detection of this cytokine in CSF would be anticipated to occur only in a situation of excessive production. IL-1β is produced by microglia/macrophages, activates other proinflammatory cytokines (IL-8 and TNF-α)(28), and up-regulates adhesion molecules that recruit neutrophils, lymphocytes, and monocytes from the blood stream(37). The IL-1 receptor antagonist reduces excitotoxic and ischemic brain injuries(11, 38), and overexpression of IL-1 receptor antagonist induced by an adenoviral vector attenuates ischemic injuries, suggesting that IL-1 is involved in the processes leading to brain injury(39). Thus, expression of IL-1β in the immature CNS(10) and, subsequently, accumulation of IL-1 in CSF may be of importance for the development of brain injury.

The CSF levels of GM-CSF and IL-10 were not significantly affected by the insult in contrast to the marked increase of these cytokines in response to stroke in adults(13). This difference may relate to the immaturity of the cytokine-mediated immune response previously recognized(15). IL-10 is an important immunoregulatory cytokine that down-regulates inflammation mediated by T lymphocytes and macrophages(40), inhibits lipopolysaccharide-induced production of TNF-α(41), suppresses the production of oxygen intermediates/nitric oxide(42), and attenuated the severity of experimental allergic encephalitis in mice(45). Therefore, a lack of IL-10 response at least in the early phase after asphyxia may aggravate brain injury.

The expression of TNF-α mRNA is markedly enhanced in the CNS after hypoxia-ischemia in adult(43) and newborn(9) rats. Nevertheless, TNF-α was detectable in only approximately 45% of CSF samples, and the levels were unaffected by the asphyxic insult in the present study, which is in accordance with results from adult stroke patients(13, 27). Cellular expression of mRNA for TNF-α may, however, not result in production of bioactive TNF-α under these conditions due to inhibition at the translational level, or locally produced TNF-α may not reach the CSF compartment due to hindrance of diffusion.

Our findings need further experimental and clinical validation before conclusions can be drawn with regard to the possible clinical significance. Analyses of cytokines may become an important adjunct to other biochemical protein markers for assessment of prognosis and for dating the injury(44). The involvement of cytokines in the cascade leading to brain injury(25) may also open up new cerebroprotective strategies in the future.