In this study, we found increased concentrations of QUIN and galectin-3 in CSF from asphyxiated infants. These increases were more pronounced in infants with severe clinical course and poor prognosis. Galectin-3 was also elevated in asphyxiated infants with abnormal outcome and the elevation remained significant within the clinically important group with moderate encephalopathy. Systemic infection/inflammation without signs of asphyxia did not result in elevated CSF concentrations.
The pathogenesis of birth asphyxia is complex and several other factors including chorioamnionitis may contribute to neonatal encephalopathy as well as perinatal brain injury [7
]. In order to define a group where perinatal asphyxia was indeed the probable cause of encephalopathy, we used well-established criteria including signs of fetal distress, neonatal need for resuscitation, and most importantly, metabolic acidosis. Previous studies show that a large proportion of encephalopathic infants fulfilling very similar criteria for asphyxia have acutely evolving injuries on magnetic resonance imaging (MRI) [38
], suggesting that the insult took place at or near the time of birth. In addition, nearly half of the asphyxiated infants in our study were exposed to severe obstetric complications that may directly impair oxygen delivery and they presented with a considerable metabolic acidosis (mean pH 6.9 and base deficit 20). In clinical intervention studies using hypothermia, eligible infants with moderate to severe encephalopathy have to meet inclusion criteria for significant birth asphyxia (pH <7, base deficit >16, low APGAR score at 10′ or prolonged need for resuscitation) [39
]. Seventeen out of 20 asphyxiated infants in our study fulfilled these criteria even if our study also included infants with mild encephalopathy. The infants with adverse outcome also met the internationally accepted clinical criteria for cerebral palsy caused by an acute intrapartum event [41
Blood gases were not routinely obtained at birth in the then apparently healthy infection and control infants. Since isolated metabolic acidosis is a common clinical finding not associated with neurological sequels in infants without encephalopathy, the control and infected infants are likely to be a true low-risk group even if early blood gases for comparisons with the asphyxiated infants were not available.
Even if the study group fulfilled strict criteria for intrapartum asphyxia, we cannot exclude that other factors including chorioamnionitis contributed to the susceptibility to asphyxia. Only one mother had clinical chorioamnionitis, but placentas were not examined and subclinical infections cannot be ruled out. However, the lack of microglia response and CSF inflammatory reaction in infants with systemic inflammation/infection makes it less likely that chorioamnionitis in itself, or the systemic inflammation seen in the asphyxiated infants, could explain the CSF changes found in infants with encephalopathy.
QUIN in CSF has previously been studied under normal as well as various pathological conditions in the pediatric population [42
], but there are no studies in newborns or after HI brain injury. The normal QUIN levels are inversely related to age with a mean concentration of 31 nM (range 20–64) in infants below 1 year of age [42
]. We report significantly higher levels in newborn control infants with a mean concentration of 116 nM. This increase may be due to the trauma of normal labor or be part of the mild inflammatory activation associated with normal birth. The latter seems less likely since infants with a pronounced systemic inflammatory response included in this study did not differ from controls. In asphyxiated infants, QUIN concentrations were elevated approximately threefold, and the concentrations were similar to those previously found in infants with intraventricular hemorrhage [43
], a clinical condition that is associated with an intense and prolonged inflammatory response in CSF [44
QUIN concentrations were also associated with a severe clinical course and poor prognosis, but the differences between asphyxiated infants with various outcomes did not reach statistical significance. QUIN concentrations may, however, be difficult to evaluate since timing is important for the absolute value. In experimental ischemia, QUIN is significantly elevated after 2 days and increases further thereafter [29
]. Serial CSF samples after trauma show an increase in QUIN concentrations up to 48 h [45
] and increased QUIN levels were associated with mortality, when corrected for time of sampling [45
]. In our study, sampling was performed at a mean of 38 h after birth in the asphyxiated group, suggesting that many infants had not reached their peak levels and that the values may not fully reflect the extent of injury. It is, however, not likely that the timing could explain the difference between groups since there was no difference in age at sampling between groups and no correlation between QUIN levels and age at sampling. In addition, there was a tendency towards later sampling in the control group suggesting that the control levels might even be overestimated in relation to the asphyxiated group.
To our knowledge, galectin-3 has not previously been measured in CSF from newborns or in association with brain injury. We report detectable levels in all samples and a significant increase in asphyxiated infants and infants with a severe clinical course, poor prognosis, and abnormal outcome. In a separate analysis of the clinically important group of infants with moderate encephalopathy, the difference in galectin-3 levels between infants with normal or abnormal outcome remained significant although the number of infants was small. Markers of abnormal outcome are of specific importance in this group since only 30–50 % of these infants develop a permanent brain injury despite similar clinical courses [1
]. We have previously shown that in the experimental setting of neonatal HI, the effect, but not the expression, of galectin-3 is gender-dependent [17
]. In this study, the groups were too small to allow for subgroup analyses, and we cannot exclude that the uneven distribution of boys and girls in the different study groups may have influenced the results.
The pattern of activation and CSF increases were similar for QUIN and galectin-3 and the concentrations in individual infants significantly correlated. This suggests that galectin-3 and QUIN are part of the same inflammatory response. The galectin-3 pattern of increase and correlation to severity of clinical course and outcome is also similar to that seen in previous studies of proinflammatory cytokines [2
MMP-9 was found in only one infant with severe asphyxia. This may be due to the detection method. Normal CSF values are below the detection level of the ELISA used [46
] and even in severe CNS inflammatory disease, MMP-9 is detected in only a minority of patients [47
An important question is whether galectin-3 and QUIN are produced within the brain or if systemic cells of the monocyte/macrophage lineage are responsible for the increase found in CSF. Galectin-3 as well as QUIN are produced by activated microglia–macrophages as part of the inflammatory response after focal cerebral insults [11
]. QUIN is produced by local de novo synthesis after focal ischemia [48
] but an increased production of QUIN within the brain is also seen in systemic inflammation [27
]. In experimental studies, systemic inflammation induced by the bacterial toxin lipopolysaccharide (LPS) results in increased CSF concentrations of QUIN [27
], but in patients with HIV, elevated serum concentrations do not correlate with CSF levels, supporting an intracerebral source of QUIN [50
]. Galectin-3 activation in focal vs. systemic inflammatory response is less well studied and it is not known whether HI or sepsis result in a systemic galectin-3 production.
Another possible mechanism by which systemically produced inflammatory mediators can contribute to elevated CSF concentration is through leakage over an injured BBB. Serum QUIN concentrations normally exceed those found in CSF [50
], while serum levels of galectin-3 under normal or inflammatory conditions are not known. In our study, CSF protein content did not differ between asphyxiated infants and controls suggesting that there were no major differences in BBB damage. The absence of correlation between CSF protein and QUIN or galectin-3 concentrations also suggests that BBB breakdown is not responsible for the increased levels seen in CSF. CSF protein level was, however, not analyzed in all samples (7/45 missing) and in the absence of simultaneous blood samples, we cannot exclude a systemic contribution. It is, however, likely that brain microglia/macrophages are an important source of CSF galectin-3 and QUIN after asphyxia. Increased expression of galectin-3 at mRNA [16
] as well as protein [17
] is seen after experimental perinatal HI and intrathecal production of QUIN with associated increases in CSF is seen in experimental brain ischemia and inflammation [29
], as well as clinical traumatic brain injury [31
Irrespective of their possible origins, both QUIN and galectin-3 have possible neurotoxic effects. QUIN is a well-known neurotoxin, acting as an NMDA receptor agonist [25
] as well as promoting oxidative injury [26
]. Inhibition of QUIN production reduces injury in adult ischemia–reperfusion injury [30
], but the immature brain has not been studied. Interestingly, studies in rats suggest that the immature brain has a specific sensitivity to QUIN with a maximal vulnerability at postnatal day 7 when the brain maturity closely resembles that seen in newborn infants [52
]. In addition, QUIN-induced ROS formation is dependent on the presence of free ferrous iron [26
]. Free iron is released after experimental HI in newborn pigs [53
] and is present in CSF from asphyxiated infants [54
]. QUIN is neurotoxic in vitro in concentrations as low as 100 nM [55
] and studies in patients with encephalitis and intracerebral production of QUIN show that QUIN concentrations in the brain is tenfold higher than that found in CSF [50
]. It is, thus, likely that brain QUIN levels in the asphyxiated patient could reach toxic levels.
Galectin-3 contributes to injury after experimental neonatal HI possibly by modulating the microglia response and MMP-9 activation [17
]. This is in stark contrast to findings in adult stroke models where galectin-3 is expressed in a microglia population with protective properties [56
] and transgenic mice lacking functional galectin-3 have aggravated injury [23
]. Galectin-3 may, thus, serve as a useful marker of severity of injury in the newborn asphyxiated infant, but further studies are needed to determine whether galectin-3 contributes to injury also in the clinical setting.
In conclusion, our study demonstrates elevated levels of macrophage/microglia-derived potentially neurotoxic inflammatory mediators in CSF from asphyxiated infants with severe clinical course and adverse outcome. These mediators have been identified in experimental studies and our study confirms their clinical relevance. In addition, the role of microglia in HI injury is under current debate and a more detailed knowledge of inflammatory mechanisms that contribute to injury or repair can, in the future, lead to targeted therapeutic interventions.