The Journal of Maternal-Fetal & Neonatal Medicine
ISSN: 1476-7058 (Print) 1476-4954 (Online) Journal homepage: http://www.tandfonline.com/loi/ijmf20
Persistent systemic monocyte and neutrophil activation in neonatal encephalopathy Fiona M. O’Hare, R. W. G. Watson, Amanda O’Neill, Alfonso Blanco, Veronica Donoghue & Eleanor J. Molloy To cite this article: Fiona M. O’Hare, R. W. G. Watson, Amanda O’Neill, Alfonso Blanco, Veronica Donoghue & Eleanor J. Molloy (2015): Persistent systemic monocyte and neutrophil activation in neonatal encephalopathy, The Journal of Maternal-Fetal & Neonatal Medicine To link to this article: http://dx.doi.org/10.3109/14767058.2014.1000294
Published online: 06 Feb 2015.
Submit your article to this journal
Article views: 32
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ijmf20 Download by: [University of Otago]
Date: 27 September 2015, At: 14:42
http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–8 ! 2015 Informa UK Ltd. DOI: 10.3109/14767058.2014.1000294
ORIGINAL ARTICLE
Persistent systemic monocyte and neutrophil activation in neonatal encephalopathy Fiona M. O’Hare1,2,3, R. W. G. Watson2, Amanda O’Neill2, Alfonso Blanco2, Veronica Donoghue1,4, and Eleanor J. Molloy1,2,5,6,7,8
Downloaded by [University of Otago] at 14:42 27 September 2015
1
Department of Paediatrics, National Maternity Hospital, Dublin, Ireland, 2UCD School of Medicine & Medical Sciences & Conway Institute of Biomolecular and Biomedical Sciences, University College Dublin, Dublin, Ireland, 3National Children’s Research Centre, Crumlin, Dublin, Ireland, 4 Department of Radiology, Children’s University Hospital, Dublin, Ireland, 5Department of Paediatrics, Royal College of Surgeons in Ireland, Dublin, Ireland, 6Department of Neonatology, Our Lady’s Children’s Hospital, Crumlin, Dublin, Ireland, 7Department of Paediatrics, Trinity College Dublin, Dublin, Ireland, and 8Academic Paediatric Centre, National Children’s Hospital, Tallaght, Dublin, Ireland Abstract
Keywords
Aim: Circulating immune cell activation is associated with worse outcome in adult and animal models of brain injury. Our aim was to profile the systemic inflammatory response over the first week of life in infants at risk of neonatal encephalopathy (NE) and correlate early neutrophil and monocyte endotoxin and activation responses with outcome. Methods: Prospective observational study in a tertiary referral university hospital including 22 infants requiring resuscitation at birth who had serial (five time points) neutrophil and monocyte CD11b (marker of cell adhesion), intracellular reactive oxygen intermediates (ROI; cell activation) and Toll-like receptor (TLR; endotoxin recognition) before and after endotoxin stimulation ex vivo compared to neonatal controls. Results: All neonates requiring resuscitation at delivery (n ¼ 122 samples) had higher neutrophil and monocyte CD11b and TLR-4 expression compared with adults and neonatal controls. Neonates with abnormal neuroimaging and/or severe NE had increased CD11b, ROI and TLR-4. Increased polymorphonuclear leukocytes TLR-4 expression was associated with increased mortality in infants with NE. Conclusion: Innate immune dysregulation in the first week of life is associated with severity of outcome in neonatal brain injury in this cohort and may be amenable to immunomodulation.
Brain injury, CD11b, immunity, reactive oxygen intermediate, Toll-like receptor 4
Introduction Perinatal asphyxia may result in neonatal encephalopathy (NE) and long-term disability. Inflammatory cells, in particular polymorphonuclear leukocytes (PMNs) and monocytes, are recruited to the central nervous system (CNS), and the ensuing tissue damage is primarily due to the host inflammatory response, as opposed to pathogenic toxins [1]. Neonatal rats rendered neutropenic have less brain swelling following hypoxia–ischaemia (HI) indicating that PMNs contribute to vascular dysfunction either during the initial insult or early hours of recovery (54–8 h) [2]. As activated systemic leukocytes are also implicated in adult stroke, we were interested in similar responses in neonates at risk of brain injury. CD11b (a b2 integrin) is a surface receptor that aids the adherence of PMNs to the endothelial cell wall, thereby facilitating their Address for correspondence: Prof. Eleanor Molloy, Consultant Neonatologist & Paediatrician, Academic Paediatric Centre, National Children’s Hospital, Tallaght, Dublin 24, Ireland. E-mail: elesean@ hotmail.com or [email protected]
History Received 4 November 2014 Accepted 16 December 2014 Published online 6 February 2015
migration to the site of injury. CD11b expression is increased on macrophages, PMNs and microglia in ischaemic areas of the brain following middle cerebral artery occlusion [3]. Systemic PMN CD11b expression is increased during reperfusion following ischaemic stroke in a mouse model [4]. The absence of adhesion molecules in knockout animals is associated with decreased cerebral infarct volume. In addition, pre-treatment with blocking antibodies to CD11b/CD18 decreases PMN accumulation and infarct size following cerebral HI [5]. Oxygen-free radicals play an important role in reperfusion/reoxygenation injury following asphyxia [6]. Reactive oxygen intermediates (ROIs) are toxic molecules released by immune cells to help to destroy invading pathogens, but overproduction of ROIs can potentially damage tissue. Neutrophils are the primary source of ROIs, although many other cell types, including monocytes, also produce ROIs albeit to a far lesser degree. ROIs have been implicated in the pathophysiology of many neurological disorders [7] and play an important role in the damage caused by cerebral ischaemia/ reperfusion [8]. In addition, PMN intracellular production of
2
F. M. O’Hare et al.
ROIs are associated with the multiple organ dysfunction syndrome seen in the adult systemic inflammatory response syndrome [9]. Toll-like receptors (TLR) are important detectors of microbial infection, and TLR-4 triggers host defence responses and lipopolysaccharide (LPS) signalling [10]. TLR signalling is thought to co-ordinate both the peripheral and CNS inflammatory responses [11]. In vivo studies demonstrate rapid induction of TLR-4 expression after ischaemia/reperfusion [12]. Mice lacking these receptors have reduced infarct size [12,13] and have improved neurological and behavioural outcomes [13]. We therefore aimed to characterise neutrophil and monocyte intracellular ROI production, CD11b and TLR-4 expression and response to LPS via serial analysis over the first week of life.
J Matern Fetal Neonatal Med, Early Online: 1–8
clinical encephalopathy recorded in the medical notes as per the classification of Sarnat & Sarnat [15] as follows: (a) NE 0/ I: infants who required resuscitation following delivery with no neurological signs or mild encephalopathy and (b) NE II/ III: moderate/severe encephalopathy. All infants had serial cranial ultrasounds performed within the first 24 h of life. Those with NE had a MRI brain within the first seven days of life. MRIs were scored and reported independently by a paediatric radiologist according to the Barkovich criteria [16], which employs a combination five-point score including components of both basal ganglia and watershed patterns of injury. Patients were divided into ‘‘normal’’ (score ¼ 0) and ‘‘abnormal’’ (score ¼ 1–4) neuroimaging groups for the purpose of data analysis. Blood sampling
Downloaded by [University of Otago] at 14:42 27 September 2015
Materials and methods Reagents The following reagents were used: LPS Escherichia coli serotype 0111:B4, foetal calf serum, dihydrorhodamine 123 (DHR 123) and phorbol 12-myristate 13-acetate (PMA) purchased from Sigma Aldrich Ireland Ltd. (Arklow, Ireland; www.sigmaaldrich.com/ireland); phycoerythrin (PE)-labelled CD11b and BD FACS lysing solution from BD Biosciences (Oxford, UK; www.bd.com/uk); Alexa Fluor 647 antihuman TLR-4 from eBioscience Ltd. (Hatfield, United Kingdom;www.eBioscience.com); Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin solution and L-glutamate from GibcoBRL Life Technologies/ Invitrogen, Co. (Dublin, Ireland; www.invitrogen.com). Study population Ethical committee approval was received from the National Maternity Hospital, Dublin, a tertiary referral, UniversityAffiliated Maternity Hospital with 49500 deliveries per annum for the study period March 2010 to March 2011. In all cases, written informed consent was taken from all parents of infants enrolled in this study. The following study groups were enrolled: Adults: Healthy adult men and non-pregnant women; neonatal controls: Umbilical cord blood following normal delivery from neonates with normal Apgar scores and postnatal course; and neonatal cases: infants with at least two of the following criteria were eligible for inclusion according to Huang et al. criteria: (i) evidence/suspicion of HI injury based on a history of foetal distress, i.e. type II dips, loss of beat-to-beat variability on cardiotocography and/or abnormal scalp pH; (ii) need for resuscitation after birth, i.e. bag and mask ventilation; (iii) base deficit 415 mmol/l or pH57.2 in cord blood or admission arterial sample [14]. Infants with congenital abnormalities or evidence of maternal substance abuse or culture-positive sepsis were excluded. The protocol commenced resuscitation in room air, and the oxygen concentration was then titrated up in 10% increments as clinically indicated at five-minute intervals. None of the infants were resuscitated in FiO2421%. On completion of this study, infants were retrospectively divided into subgroups according to the highest grade of
Neonatal blood sampling at 0–24, 24–48, 48–72, 72–96 h and day 7 of life, was paired with routine phlebotomy following fully informed consent from parents. Arterial samples were taken when peripheral or umbilical arterial catheters were in situ, otherwise peripheral venous samples were obtained. At each time point, 1 mL was collected in sodium citrate anticoagulated (whole blood) bottles. Whole blood samples were kept in ice and processed within 90 min and was incubated for one hour in 37 C with pro-inflammatory agent LPS 1 mg/mL to mimic an inflammatory response in vitro [17]. Quantification of respiratory burst activity Generation of ROI was evaluated by flow cytometry using the technique of Smith and Wiedemann [18]. Whole blood (50 mL) was incubated ± LPS (1 mL) at 37 C for 1 h. All samples were subsequently incubated with DHR (100 mM) at 37 C for 10 min before stimulation with 1 mL (16 mM) of PMA for 20 min at 37 C. The reaction was then halted by placing samples on ice. Samples were analyzed using an Accuri C6 flow cytometer with CFlow Plus software (BD Biosciences, Oxford, United Kingdom). Leukocyte populations were selected based on their scatter profiles, forward scatter and side scatter. Neutrophil and monocyte ROI fluorescence intensity was collected on the photomultiplier 2 (FL-2-A) using a 585/40 filter and expressed as mean channel fluorescence. Each sample was acquired over 2 min at medium speed. DHR detects mainly intracellular H2O2 and OH radical production [18]. Quantification of cell surface antigen expression The expression of CD11b and TLR-4 antigens on the surface of neutrophils and monocytes was measured by flow cytometry. Whole blood (50 mL) was treated with 5 mL PE-CD11b and 2.5 mL anti-human TLR-4 antibody and left at 4 C for 20 min. FACS was added and incubated for 10 min at room temperature. The sample was centrifuged at 3000 rpm for five minutes at 4 C. The pellet was suspended twice with DMEM 500 mL and stored on ice before analysis by flow cytometry. Each sample was acquired over two minutes at medium speed, and a minimum of 5000 events were collected and analyzed under the same instrument settings [19].
Neonatal brain injury
DOI: 10.3109/14767058.2014.1000294
Statistics Statistical analysis was carried out using analysis of variance (ANOVA) using PASW statistical package version 18 (Armonk, NY; www.ibm.com/SPSS_Statistics). Two-way ANOVA was used in the comparison of baseline and LPSinduced CD11b and TLR-4 expression between neonates and adults. Equal variance was assumed and Tukey’s post hoc multiple comparisons was used. Chi squared statistic and independent samples t-test were carried out for analysis of demographics. Significance was assumed for values of p50.05. Results are expressed as mean ± standard error of the mean (SEM) unless otherwise indicated.
Results
Downloaded by [University of Otago] at 14:42 27 September 2015
Clinical characteristics There were 122 samples from 22 newborn infants who required resuscitation at delivery (resuscitation only: NE 0: n ¼ 4; NE I: n ¼ 4; NE II: n ¼ 9; and NE III: n ¼ 5). This was a convenience sample taken when FM was available to process the samples. Ten infants were eligible for therapeutic hypothermia, in accordance with the Total Body Hypothermia for Neonatal Encephalopathy criteria [20], and were treated for 72 h duration. Six infants died. There were no differences in gender distribution, birth weight, mode of delivery, Apgar scores, cord pH or admission blood gas parameters between normal and abnormal neuroimaging groups (Table 1). Eighteen infants had a MRI brain in the first week of life with normal imaging (n ¼ 5; score ¼ 0); abnormal signal in the basal ganglia (n ¼ 1; score ¼ 1), abnormal cortical signal (n ¼ 5; score ¼ 2); abnormal signal in both the cortex and basal ganglia/thalamus (n ¼ 2) and widespread abnormal signal involving the entire cortex and basal ganglia/thalamus (n ¼ 5). The remainder had a cranial ultrasound only, which was normal in all cases. CD11b surface expression and NE All neonates, irrespective of insult severity, demonstrated higher basal PMN CD11b expression compared to adults, which was significant in the NE 0/I group on day 3 of life
(p ¼ 0.038) and in NE II/III group at birth, day 4 and day 7 of life (p50.001, p ¼ 0.014 and p ¼ 0.013, respectively; Figure 1a). Following in vitro LPS stimulation, infants with NE had increased surface expression of CD11b to a greater extent than adults. This was significant in both groups at birth (NE 0/I: p ¼ 0.043; NE II/III: p ¼ 0.022) and in the NE II/III group on day 2 of life (p ¼ 0.032; Figure 1b). All NE infants expressed significantly higher monocyte baseline and post LPS stimulation levels of CD11b compared to neonatal controls from birth to 96 h of life. This significantly higher expression level persisted to day 7 of life in the NE II/III group (Figure 1c and d). Higher baseline monocyte CD11b expression was seen in all neonates compared to adults throughout the first week of life, which was statistically significant in NE 0/I neonates on day 3 (p ¼ 0.015) and in NE II/III neonates from birth to day four (0–24 h: p ¼ 0.002; 24–48 h: p ¼ 0.003; 48–72 h: p ¼ 0.007; 72–96 h: p ¼ 0.002; Figure 1c). Neonates with NE II/III displayed significantly elevated post-stimulation CD11b levels compared to adults on day 4 (p ¼ 0.018; Figure 1d). Higher PMN CD11b expression was demonstrated in the abnormal neuroimaging (AI) group and following stimulation with LPS in both neonatal groups at 0–24 h compared to adults. Monocyte CD11b expression was significantly increased at baseline in (a) both neonatal groups from birth to 96 h of life compared to adults and (b) abnormal neuroimaging group compared with neonatal controls on day 7. Following LPS stimulation, CD11b expression was significantly greater tin both neonatal groups compared to neonatal controls from birth to 96 h of life. This persisted in the abnormal neuroimaging group on day 7. Neutrophil and monocyte ROI production is increased in NE All newborns requiring resuscitation at delivery had higher ROI production at baseline in the first 48 h compared to adults and neonatal controls, irrespective of insult severity. Newborns with NE 0/I had comparable ROI levels to adults and neonatal controls thereafter (Figure 2a). Upregulation of ROI production, following LPS stimulation, occurred to a greater magnitude in newborns than in
Table 1. Demographics of infants with neonatal encephalopathy divided by normal versus abnormal neuroimaging.
Male sex (%) GA BW (g) SVD (%) Pre-L Em (%) In-L Em (%) Apgar 1 min Apgar 5 min Cord pH Cord Bxs Admission pH Admission Bxs Admission lactate
3
Normal neuroimaging
Abnormal neuroimaging
p value
64 39+5 (2+0) 3827 (575) 50 43 57 3 (1) 5 (2) 7.05 (0.22) 12.2 (8.4) 7.06 (0.26) 10.4 (11.7) 10.1 (5.7)
56 40+6 (1+0) 3487 (575) 22 0 100 3 (3) 5 (3) 6.97 (0.13) 14.3 (4.0) 7.08 (0.28) 15.3 (10.2) 16.9
0.505 0.133 0.180 0.187 NA NA 0.968 0.738 0.234 0.506 0.860 0.301 0.367
GA, gestational age; BW, birth weight; SVD, spontaneous vaginal delivery; Pre-L Em, prelabour emergency caesarean section; In-L Em, in labour emergency caesarean section; and BE, base excess.
Downloaded by [University of Otago] at 14:42 27 September 2015
4
F. M. O’Hare et al.
J Matern Fetal Neonatal Med, Early Online: 1–8
Figure 1. PMN and monocyte CD11b expression and encephalopathy grade neutrophil and monocyte CD11b expression assessed in healthy adult controls (dark grey), neonatal controls (light grey) (umbilical cord blood), infants requiring resuscitation at birth with no/mild neurological abnormalities (white) (NE 0/I) or moderate/severe abnormality or death (black) (NE II/III) at baseline (a and c) and following LPS stimulation (b and d). *p50.05 versus adult controls, yp50.05 versus neonatal controls. Results expressed as mean channel fluorescence (MCF).
adults or neonatal controls (Figure 2b). Newborns with NE II/ III produced significantly greater ROIs compared to NE 0/I at 72–96 h (p ¼ 0.036) and day 7 of life (p ¼ 0.035; Figure 2b). Monocytes produce 80% less ROIs than neutrophils under the same conditions both at baseline and following stimulation. Neonatal monocytes in both groups produced significantly greater quantities of ROI in the first 48 h of life compared to adult monocytes [NE 0/I: p ¼ 0.001 (0–24 h), p ¼ 0.012 (24–48 h); NE II/III: p50.001, at 0–24 and 24– 48 h, respectively)]. Monocyte ROI production remained significantly higher in infants with NE II/III compared to adults throughout the first week of life. ROI production was higher in NE II/III compared to NE 0/I from birth to day 7 of life and reached statistically significant levels at 48–72 h of life (p ¼ 0.001). All neonates requiring resuscitation at delivery had significantly higher baseline ROI production at birth compared to neonatal controls (NE 0/I: p ¼ 0.012; NE II/ III: p50.001). This elevated ROI production persisted to day 7 of life in the NE II/III group (Figure 2c). Monocyte ROI production was significantly increased following in vitro LPS stimulation in all newborns compared to adults from birth to 48 h of life (NE 0/I: p ¼ 0.019 (0–24 h), p ¼ 0.003 (24–48 h); NE II/III NE: p50.001, at 0–24 and 24–48 h, respectively). Post stimulation, ROI production remained significantly increased in NE II/III compared to adults through to day 7 of life. Both neonatal NE groups had higher post LPS stimulation ROI production compared with neonatal controls.
This reached statistical significance from birth to day 4 of life in the NE II/III group (p50.001 at all time points) and at 24– 48 h in the NE 0/I group (p ¼ 0.015). Greater ROI production was noted in NE II/III monocytes, following LPS stimulation, compared to NE 0/I at 48–72 h (p ¼ 0.002) and 72–96 h of life (p ¼ 0.036; Figure 2d). LPS hyporesponsiveness was initially displayed by the abnormal compared to the normal neuroimaging group with a 5% versus 35% increase in ROI production following LPS stimulation. We also demonstrated an association between ROI production over the first week of life and abnormalities on neuroimaging. Infants requiring resuscitation at delivery with subsequent neuroimaging abnormalities demonstrated increased ROI production compared to infants with normal neuroimaging, especially from day 3 of life onwards (72–96 h; p ¼ 0.060; day 7, p ¼ 0.052; data not shown). LPS hyporesponsiveness was initially found in the abnormal neuroimaging group compared to the normal neuroimaging group (5% versus 35% increase in ROI production following LPS stimulation). Treatment with therapeutic hypothermia (33–34 C for 72 h) had no demonstrable reduction in neutrophil or monocyte ROI production either at baseline or following LPS stimulation. Monocyte ROI production was increased from birth to 48 h of life in all infants requiring resuscitation at delivery, both at baseline and following LPS stimulation, compared to 2 adults. Neutrophil ROI production remained
Neonatal brain injury
Downloaded by [University of Otago] at 14:42 27 September 2015
DOI: 10.3109/14767058.2014.1000294
5
Figure 2. PMN and monocyte ROI production and encephalopathy grade neutrophil and monocyte ROI production assessed in healthy adult controls (dark grey), neonatal controls (light grey) (umbilical cord blood), infants requiring resuscitation at birth with no/mild neurological abnormalities (white) (NE 0/I) or moderate/severe abnormality or death (black) (NE II/III) at baseline (a and c) and following LPS stimulation (b and d)). (a) *p50.05 versus adult controls. (b) *p50.05 versus adult controls, yp50.05 NE 0/I versus NE II/II. (c) *p50.05 versus adult controls, yp50.05 versus neonatal controls. (d) *p50.05 versus adult controls, yp50.05 versus neonatal controls, zp50.05 NE 0/I versus NE II/III. Results expressed as mean channel fluorescence (MCF).
elevated in infants who received therapeutic hypothermia for 72 h compared to adults up to day 7 of life. TLR-4 surface expression and NE There was a significant increase in LPS-stimulated neutrophil TLR-4 in NE non-survivors compared to neonatal controls (p ¼ 0.04). On day 7, there was a significant difference between baseline TLR-4 neonatal controls versus NE survivors (p ¼ 0.031) and stimulated TLR-4 in non-survivors versus survivors (p ¼ 0.044) (data not shown). Baseline PMN TLR-4 expression was significantly increased in both NE groups, compared to adults, at all time points from birth to day 7 of life (Figure 3a). Post LPS stimulation, PMN TLR-4 expression was significantly increased in both NE groups, compared to adults, from birth to day four of life and remained significantly elevated in the NE II/III group on day 7 of life (Figures 3b). Baseline monocyte TLR-4 expression was significantly increased in neonates with NE 0/I compared to adults from birth to 72 h of life and in the NE II/III group from birth to day 7 of life (Figure 3c). All neonates, irrespective of the degree of encephalopathy, expressed higher levels of monocyte TLR-4 following LPS stimulation compared to adults. This was significant in NE II/ III neonates from birth to day 7 of life (0–24 h: p ¼ 0.037; 24– 48 h: p ¼ 0.003; 48–72 h: p ¼ 0.002; 72–96 h: p ¼ 0.001; day 7: p ¼ 0.041; Figure 3d). Basal TLR-4 expression in neonatal
monocytes was significantly elevated at birth compared to adults but not neonatal controls. Therapeutic hypothermia did not significantly alter PMN or monocyte CD11b or TLR-4 expression at any time point during the first week of life.
Discussion We have shown that term neonates following resuscitation at birth have a vigorous systemic innate immune response compared to adults and neonatal controls over the first week of life. Neonates with more severe encephalopathy grades (NE II/III) produced significantly higher basal PMN CD11b levels on days 1, 4 and 7 and monocyte CD11b expression from days 1–4 of life. Neonatal monocyte CD11b expression, irrespective of neuroimaging outcome or encephalopathy grade, was significantly higher than neonatal controls from birth to 96 h of life and remained significantly elevated in the abnormal neuroimaging and NE II/III groups on day 7 of life. Delayed PMN apoptosis with increased CD18/CD11b expression and interleukin (IL)-8 (a PMN chemoattractant) secretion (following LPS stimulation) have been described in neonates suggesting their potential role in neonatal inflammation [21,22]. We have [23] previously reported increased cord PMN CD11b expression in neonates requiring resuscitation at birth with normal neurological outcome and suppression of PMN CD11b expression in neonates with severe neurological signs. Hashimoto et al. [24] found reduced neutrophil
Downloaded by [University of Otago] at 14:42 27 September 2015
6
F. M. O’Hare et al.
J Matern Fetal Neonatal Med, Early Online: 1–8
Figure 3. PMN and monocyte TLR4 expression and encephalopathy grade neutrophil and monocyte TLR-4 expression assessed in healthy adult controls (dark grey), neonatal controls (light grey) (umbilical cord blood), infants requiring resuscitation at birth with no/mild neurological abnormalities (white) (NE 0/I) or moderate/severe abnormality or death (black) (NE II/III) at baseline (a and c) and following LPS stimulation (b and d). *p50.05 versus adult controls. Results expressed as mean channel fluorescence (MCF).
expression of the adhesion molecule L-selectin (shed prior to neutrophil infiltration) in peripheral blood samples from neonates with severe asphyxia within two hours of birth, but did not show any significant differences in PMN CD11b expression between normal neonates and those with mild or severe asphyxia. PMN and monocyte TLR-4 expression was significantly greater than adult levels in the NE II/III group from birth to day 7 of life. Following in vitro LPS stimulation, neonatal PMNs and monocytes upregulated expression to a greater magnitude than adult PMNs. Although TLR-4 expression is increased on adult isolated PMNs under hypoxic conditions, neonatal cord PMNs do not increase TLR-4 expression in vitro [19]. TLR-2 and 4 mRNA is decreased in cord blood mononuclear cells from infants with asphyxia [25]. Animal studies report improved neurological outcomes in TLR-4deficient mice following HI injury and also intracerebral haemorrhage [26]. Increased monocyte TLR-4 correlates with severity of acute cerebral infarction in adults [27]. Specific TLR-4 antagonists decrease brain endothelial activation and neutrophil transmigration in vitro [28]. Persistent inflammation may be associated with persistent activation of hypoxia-inducible factor (HIF) 1 alpha [29]. In addition, upregulated HIF-1a is associated with enhanced antibactericidal activity and phagocytosis and persistent neutrophilic inflammation [30]. In lung ischaemia/reperfusion injury, HIF-1a protein upregulates TLR4 expression in a positive feedback manner [31]. HIF-1a antibody
neutralization attenuates the increase of TLR4 expression in hypoxic microglial cells [32]. TLR4 expression in macrophages is upregulated via HIF-1 in response to hypoxia [33]. Delayed or persistent inflammatory responses have been demonstrated by other groups including a delayed rise in Creactive protein in babies undergoing hypothermia therapy [34]. Youn et al. [35] found persistent elevation of IL-10 and IL-8 in infants with NE and seizures. Vascular endothelial growth factor, glial fibrillary protein and interferon-gamma were persistently elevated in moderate/severe HIE compared to infants with mild HIE at 78–96 h [36]. Advances in neuroimaging techniques have demonstrated that brain injury evolves over days and weeks [37]. Persistence of systemic inflammation was demonstrated as increased systemic ROI production in newborns requiring resuscitation at delivery, which was increased in severe NE. Neutrophil ROI production in adults is decreased in the setting of in vitro and in vivo hypothermia [38]. Therapeutic hypothermia did not significantly alter neutrophil or monocyte ROI production, either at baseline or following LPS stimulation, in our patient population. Other research groups describe a reduction in systemic serum-free radical production in the setting of hypothermia [39] both in vivo and in vitro studies [38], but no other group has studied intracellular ROI production in monocytes and neutrophils, making comparison with other groups difficult. Blocking neutrophil ROI production is a potential target for adjunctive therapy. Many different agents including dexamethasone, IL-10, vitamin A derivatives, vitamin D,
Downloaded by [University of Otago] at 14:42 27 September 2015
DOI: 10.3109/14767058.2014.1000294
vitamin E, uric acid, minocycline and NADPH oxidase inhibition [40] have potential roles as modulators of oxidantmediated injury and decrease ROI production in in vitro studies. This study has several limitations, as comparisons with other studies, which only analysed umbilical cord blood at a single time point, is difficult. Many studies have shown major disparities between cord blood and postnatal neonatal blood inflammatory profiles especially as cord blood is relatively endotoxin tolerant [41]. To our knowledge, there are no human studies to date of TLR-4, CD11b expression and intracellular ROI production in neonatal NE published. Previously Vento et al. have demonstrated increased serum oxidant levels in asphyxiated infants resuscitated with FiO2 100% compared to room air [42]. In this study, we did not measure serum oxidants as several studies have shown a poor correlation with neutrophil intracellular ROIs, and we have shown LPS hyporesponsiveness of neonatal neutrophils compared with adults in hypoxia [43,44]. We were interested in neutrophil and monocyte activation and therefore concentrated on intracellular ROI production. NE remains a significant cause of long-term morbidity and mortality. At present, there are no universally accepted individual markers to pinpoint those asphyxiated infants at highest risk of significant neurological sequelae, although multiple associations have been studied. Further validation of these findings will allow the development of early tests to predict outcome and counsel families. Understanding the immune response in severely affected babies with NE will also allow development of immunomodulatory adjunctive therapies.
Acknowledgements We thank Dr John Murphy, Dr Anne Twomey, Dr Colm O’Donnell and Dr Deirdre Sweetman for all their support and assistance in recruiting patients for this study. We wish to thank all of the parents, babies, laboratory and hospital staff who generously participated in this project. In addition, we wish to thank Dr Bryan Lynch for his expert Neurology opinion and Professor Billy Bourke for his support and encouragement.
Declaration of interest The authors have no further disclosures or conflicts of interest. This study was funded by the National Children’s Research Centre, Crumlin, Dublin 12, Ireland and the National Maternity Hospital Fund, Holles Street, Dublin 2, Ireland.
References 1. Nathan C, Ding A. Non-resolving inflammation. Cell 2010;140: 871–82. 2. Palmer C, Roberts RL, Young PI. Timing of neutrophil depletion influences long-term neuroprotection in neonatal rat hypoxicischemic brain injury. Pediatr Res 2004;55:549–56. 3. Kim JS, Chopp M, Chen H, et al. Adhesive glycoproteins CD11a and CD18 are upregulated in the leukocytes from patients with ischemic stroke and transient ischemic attacks. J Neurol Sci 1995; 128:45–50.
Neonatal brain injury
7
4. Morrison H, McKee D, Ritter L. Systemic neutrophil activation in a mouse model of ischemic stroke and reperfusion. Biol Res Nurs 2011;13:154–63. 5. Matsuo Y, Onodera H, Shiga Y, et al. Role of cell adhesion molecules in brain injury after transient middle cerebral artery occlusion in the rat. Brain Res 1994;656:344–52. 6. Kumar A, Ramakrishna SV, Basu S, Rao GR. Oxidative stress in perinatal asphyxia. Pediatr Neurol 2008;38:181–5. 7. Siesjo BK, Agardh CD, Bengtsson F. Free radicals and brain damage. Cerebrovasc Brain Metab Rev 1989;1:165–211. 8. Chan PH. Role of oxidants in ischemic brain damage. Stroke 1996; 27:1124–9. 9. Jimenez MF, Watson RW, Parodo J, et al. Dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome. Arch Surg 1997;132:1263–9; discussion 1269–70. 10. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5:987–95. 11. Downes CE, Crack PJ. Neural injury following stroke: are Toll-like receptors the link between the immune system and the CNS? Br J Pharmacol 2010;160:1872–88. 12. Tang SC, Arumugam TV, Xu X, et al. Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci USA 2007;104:13798–803. 13. Cao CX, Yang QW, Lv FL, et al. Reduced cerebral ischemiareperfusion injury in Toll-like receptor 4 deficient mice. Biochem Biophys Res Commun 2007;353:509–14. 14. Huang CC, Wang ST, Chang YC, et al. Measurement of the urinary lactate: creatinine ratio for the early identification of newborn infants at risk for hypoxic-ischemic encephalopathy. N Engl J Med 1999;341:328–35. 15. Sarnat HB, Sarnat MS. Neonatal encephalopathy following fetal distress. A clinical and electroencephalographic study. Arch Neurol 1976;33:696–705. 16. Barkovich AJ, Hajnal BL, Vigneron D, et al. Prediction of neuromotor outcome in perinatal asphyxia: evaluation of MR scoring systems. AJNR Am J Neuroradiol 1998;19:143–9. 17. Molloy EJ, O’Neill AJ, Grantham JJ, et al. Labor promotes neonatal neutrophil survival and lipopolysaccharide responsiveness. Pediatr Res 2004;56:99–103. 18. Smith JA, Weidemann MJ. Further characterization of the neutrophil oxidative burst by flow cytometry. J Immunol Methods 1993; 162:261–8. 19. Molloy EJ, O’Neill AJ, Doyle BT, et al. Effects of heat shock and hypoxia on neonatal neutrophil lipopolysaccharide responses: altered apoptosis, Toll-like receptor-4 and CD11b expression compared with adults. Biol Neonate 2006;90:34–9. 20. Azzopardi DV, Strohm B, Edwards AD, et al. Moderate hypothermia to treat perinatal asphyxial encephalopathy. N Engl J Med 2009;361:1349–58. 21. Koenig JM, Stegner JJ, Schmeck AC, et al. Neonatal neutrophils with prolonged survival exhibit enhanced inflammatory and cytotoxic responsiveness. Pediatr Res 2005;57:424–9. 22. Nguyen CN, Schnulle PM, Chegini N, et al. Neonatal neutrophils with prolonged survival secrete mediators associated with chronic inflammation. Neonatology 2010;98:341–7. 23. Molloy EJ, O’Neill AJ, Grantham-Sloan JJ, et al. Neonatal encephalopathy is associated with altered perinatal systemic neutrophil apoptosis. Am J Perinatol 2007;24:525–30. 24. Hashimoto M, Nishida A, Minakami H, et al. Decreased expression of L-selectin on peripheral blood polymorphonuclear leukocytes in neonates with severe asphyxia. Biol Neonate 2002;81: 95–8. 25. Wang L, Xu JB, Tian Y, et al. Expression and significance of Tolllike receptors in cord blood mononuclear cells. Zhonghua Er Ke Za Zhi 2007;45:365–8. 26. Sansing LH, Harris TH, Welsh FA, et al. Toll-like receptor 4 contributes to poor outcome after intracerebral hemorrhage. Ann Neurol 2011;70:646–56. 27. Yang QW, Li JC, Lu FL, et al. Upregulated expression of toll-like receptor 4 in monocytes correlates with severity of acute cerebral infarction. J Cereb Blood Flow Metab 2008;28:1588–96. 28. Leow-Dyke S, Allen C, Denes A, et al. Neuronal toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro. J Neuroinflammation 2012;9: 230.
Downloaded by [University of Otago] at 14:42 27 September 2015
8
F. M. O’Hare et al.
29. Anand RJ, Gribar SC, Li J, et al. Hypoxia causes an increase in phagocytosis by macrophages in a HIF-1alpha-dependent manner. J Leukoc Biol 2007;82:1257–65. 30. McGovern NN, Cowburn AS, Porter L, et al. Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils. J Immunol 2011;186:453–63. 31. Zhou Z, Zhu X, Chen J, et al. The interaction between Tolllike receptor 4 signaling pathway and hypoxia-inducible factor 1a in lung ischemia-reperfusion injury. J Surg Res 2014;188: 290–7. 32. Yao L, Kan EM, Lu J, et al. Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia. J Neuroinflammation 2013;10:23. doi:10.1186/1742-2094-10-23. 33. Kim SY, Choi YJ, Joung SM, et al. Hypoxic stress up-regulates the expression of Toll-like receptor 4 in macrophages via hypoxiainducible factor. Immunology 2010;129:516–24. 34. Chakkarapani E, Davis J, Thoresen M. Therapeutic hypothermia delays the C-reactive protein response and suppresses white blood cell and platelet count in infants with neonatal encephalopathy. Arch Dis Child Fetal Neonatal Ed 2014;99:F458–63. 35. Youn YA, Kim SJ, Sung IK, et al. Serial examination of serum IL-8, IL-10 and IL-1Ra levels is significant in neonatal seizures induced by hypoxic-ischaemic encephalopathy. Scand J Immunol 2012;76:286–93. 36. Chalak LF, Sa´nchez PJ, Adams-Huet B, et al. Biomarkers for severity of neonatal hypoxic-ischemic encephalopathy and
J Matern Fetal Neonatal Med, Early Online: 1–8
37. 38. 39. 40. 41.
42. 43.
44.
outcomes in newborns receiving hypothermia therapy. J Pediatr 2014;164:468–74. McKinstry RC, Miller JH, Snyder AZ, et al. A prospective, longitudinal diffusion tensor imaging study of brain injury in newborns. Neurology 2002;59:824–33. Wenisch C, Narzt E, Sessler DI, et al. Mild intraoperative hypothermia reduces production of reactive oxygen intermediates by polymorphonuclear leukocytes. Anesth Analg 1996;82:810–16. Globus MY, Alonso O, Dietrich WD, et al. Glutamate release and free radical production following brain injury: effects of posttraumatic hypothermia. J Neurochem 1995;65:1704–11. Chen H, Kim GS, Okami N, et al. NADPH oxidase is involved in post-ischemic brain inflammation. Neurobiol Dis 2011;42:341–8. Gessler P, Neu S, Nebe T, Speer CP. Granulocyte colonystimulating factor receptor expression on neutrophils of term and preterm neonates with and without signs of infection. Eur J Pediatr 1999;158:497–500. Vento M, Asensi M, Sastre J, et al. Oxidative stress in asphyxiated term infants resuscitated with 100% oxygen. J Pediatr 2003;142: 240–6. Maor I, Rainis T, Lanir A, Lavy A. Oxidative stress, inflammation and neutrophil superoxide release in patients with Crohn’s disease: distinction between active and non-active disease. Dig Dis Sci 2008;53:2208–14. Molloy EJ, O’Neill AJ, Doyle BT, et al. Effects of heat shock and hypoxia on neonatal neutrophil lipopolysaccharide responses: altered apoptosis, Toll-like receptor-4 and CD11b expression compared with adults. Biol Neonate 2006;90:34–9.
ISSN: 1476-7058 (Print) 1476-4954 (Online) Journal homepage: http://www.tandfonline.com/loi/ijmf20
Persistent systemic monocyte and neutrophil activation in neonatal encephalopathy Fiona M. O’Hare, R. W. G. Watson, Amanda O’Neill, Alfonso Blanco, Veronica Donoghue & Eleanor J. Molloy To cite this article: Fiona M. O’Hare, R. W. G. Watson, Amanda O’Neill, Alfonso Blanco, Veronica Donoghue & Eleanor J. Molloy (2015): Persistent systemic monocyte and neutrophil activation in neonatal encephalopathy, The Journal of Maternal-Fetal & Neonatal Medicine To link to this article: http://dx.doi.org/10.3109/14767058.2014.1000294
Published online: 06 Feb 2015.
Submit your article to this journal
Article views: 32
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ijmf20 Download by: [University of Otago]
Date: 27 September 2015, At: 14:42
http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–8 ! 2015 Informa UK Ltd. DOI: 10.3109/14767058.2014.1000294
ORIGINAL ARTICLE
Persistent systemic monocyte and neutrophil activation in neonatal encephalopathy Fiona M. O’Hare1,2,3, R. W. G. Watson2, Amanda O’Neill2, Alfonso Blanco2, Veronica Donoghue1,4, and Eleanor J. Molloy1,2,5,6,7,8
Downloaded by [University of Otago] at 14:42 27 September 2015
1
Department of Paediatrics, National Maternity Hospital, Dublin, Ireland, 2UCD School of Medicine & Medical Sciences & Conway Institute of Biomolecular and Biomedical Sciences, University College Dublin, Dublin, Ireland, 3National Children’s Research Centre, Crumlin, Dublin, Ireland, 4 Department of Radiology, Children’s University Hospital, Dublin, Ireland, 5Department of Paediatrics, Royal College of Surgeons in Ireland, Dublin, Ireland, 6Department of Neonatology, Our Lady’s Children’s Hospital, Crumlin, Dublin, Ireland, 7Department of Paediatrics, Trinity College Dublin, Dublin, Ireland, and 8Academic Paediatric Centre, National Children’s Hospital, Tallaght, Dublin, Ireland Abstract
Keywords
Aim: Circulating immune cell activation is associated with worse outcome in adult and animal models of brain injury. Our aim was to profile the systemic inflammatory response over the first week of life in infants at risk of neonatal encephalopathy (NE) and correlate early neutrophil and monocyte endotoxin and activation responses with outcome. Methods: Prospective observational study in a tertiary referral university hospital including 22 infants requiring resuscitation at birth who had serial (five time points) neutrophil and monocyte CD11b (marker of cell adhesion), intracellular reactive oxygen intermediates (ROI; cell activation) and Toll-like receptor (TLR; endotoxin recognition) before and after endotoxin stimulation ex vivo compared to neonatal controls. Results: All neonates requiring resuscitation at delivery (n ¼ 122 samples) had higher neutrophil and monocyte CD11b and TLR-4 expression compared with adults and neonatal controls. Neonates with abnormal neuroimaging and/or severe NE had increased CD11b, ROI and TLR-4. Increased polymorphonuclear leukocytes TLR-4 expression was associated with increased mortality in infants with NE. Conclusion: Innate immune dysregulation in the first week of life is associated with severity of outcome in neonatal brain injury in this cohort and may be amenable to immunomodulation.
Brain injury, CD11b, immunity, reactive oxygen intermediate, Toll-like receptor 4
Introduction Perinatal asphyxia may result in neonatal encephalopathy (NE) and long-term disability. Inflammatory cells, in particular polymorphonuclear leukocytes (PMNs) and monocytes, are recruited to the central nervous system (CNS), and the ensuing tissue damage is primarily due to the host inflammatory response, as opposed to pathogenic toxins [1]. Neonatal rats rendered neutropenic have less brain swelling following hypoxia–ischaemia (HI) indicating that PMNs contribute to vascular dysfunction either during the initial insult or early hours of recovery (54–8 h) [2]. As activated systemic leukocytes are also implicated in adult stroke, we were interested in similar responses in neonates at risk of brain injury. CD11b (a b2 integrin) is a surface receptor that aids the adherence of PMNs to the endothelial cell wall, thereby facilitating their Address for correspondence: Prof. Eleanor Molloy, Consultant Neonatologist & Paediatrician, Academic Paediatric Centre, National Children’s Hospital, Tallaght, Dublin 24, Ireland. E-mail: elesean@ hotmail.com or [email protected]
History Received 4 November 2014 Accepted 16 December 2014 Published online 6 February 2015
migration to the site of injury. CD11b expression is increased on macrophages, PMNs and microglia in ischaemic areas of the brain following middle cerebral artery occlusion [3]. Systemic PMN CD11b expression is increased during reperfusion following ischaemic stroke in a mouse model [4]. The absence of adhesion molecules in knockout animals is associated with decreased cerebral infarct volume. In addition, pre-treatment with blocking antibodies to CD11b/CD18 decreases PMN accumulation and infarct size following cerebral HI [5]. Oxygen-free radicals play an important role in reperfusion/reoxygenation injury following asphyxia [6]. Reactive oxygen intermediates (ROIs) are toxic molecules released by immune cells to help to destroy invading pathogens, but overproduction of ROIs can potentially damage tissue. Neutrophils are the primary source of ROIs, although many other cell types, including monocytes, also produce ROIs albeit to a far lesser degree. ROIs have been implicated in the pathophysiology of many neurological disorders [7] and play an important role in the damage caused by cerebral ischaemia/ reperfusion [8]. In addition, PMN intracellular production of
2
F. M. O’Hare et al.
ROIs are associated with the multiple organ dysfunction syndrome seen in the adult systemic inflammatory response syndrome [9]. Toll-like receptors (TLR) are important detectors of microbial infection, and TLR-4 triggers host defence responses and lipopolysaccharide (LPS) signalling [10]. TLR signalling is thought to co-ordinate both the peripheral and CNS inflammatory responses [11]. In vivo studies demonstrate rapid induction of TLR-4 expression after ischaemia/reperfusion [12]. Mice lacking these receptors have reduced infarct size [12,13] and have improved neurological and behavioural outcomes [13]. We therefore aimed to characterise neutrophil and monocyte intracellular ROI production, CD11b and TLR-4 expression and response to LPS via serial analysis over the first week of life.
J Matern Fetal Neonatal Med, Early Online: 1–8
clinical encephalopathy recorded in the medical notes as per the classification of Sarnat & Sarnat [15] as follows: (a) NE 0/ I: infants who required resuscitation following delivery with no neurological signs or mild encephalopathy and (b) NE II/ III: moderate/severe encephalopathy. All infants had serial cranial ultrasounds performed within the first 24 h of life. Those with NE had a MRI brain within the first seven days of life. MRIs were scored and reported independently by a paediatric radiologist according to the Barkovich criteria [16], which employs a combination five-point score including components of both basal ganglia and watershed patterns of injury. Patients were divided into ‘‘normal’’ (score ¼ 0) and ‘‘abnormal’’ (score ¼ 1–4) neuroimaging groups for the purpose of data analysis. Blood sampling
Downloaded by [University of Otago] at 14:42 27 September 2015
Materials and methods Reagents The following reagents were used: LPS Escherichia coli serotype 0111:B4, foetal calf serum, dihydrorhodamine 123 (DHR 123) and phorbol 12-myristate 13-acetate (PMA) purchased from Sigma Aldrich Ireland Ltd. (Arklow, Ireland; www.sigmaaldrich.com/ireland); phycoerythrin (PE)-labelled CD11b and BD FACS lysing solution from BD Biosciences (Oxford, UK; www.bd.com/uk); Alexa Fluor 647 antihuman TLR-4 from eBioscience Ltd. (Hatfield, United Kingdom;www.eBioscience.com); Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin solution and L-glutamate from GibcoBRL Life Technologies/ Invitrogen, Co. (Dublin, Ireland; www.invitrogen.com). Study population Ethical committee approval was received from the National Maternity Hospital, Dublin, a tertiary referral, UniversityAffiliated Maternity Hospital with 49500 deliveries per annum for the study period March 2010 to March 2011. In all cases, written informed consent was taken from all parents of infants enrolled in this study. The following study groups were enrolled: Adults: Healthy adult men and non-pregnant women; neonatal controls: Umbilical cord blood following normal delivery from neonates with normal Apgar scores and postnatal course; and neonatal cases: infants with at least two of the following criteria were eligible for inclusion according to Huang et al. criteria: (i) evidence/suspicion of HI injury based on a history of foetal distress, i.e. type II dips, loss of beat-to-beat variability on cardiotocography and/or abnormal scalp pH; (ii) need for resuscitation after birth, i.e. bag and mask ventilation; (iii) base deficit 415 mmol/l or pH57.2 in cord blood or admission arterial sample [14]. Infants with congenital abnormalities or evidence of maternal substance abuse or culture-positive sepsis were excluded. The protocol commenced resuscitation in room air, and the oxygen concentration was then titrated up in 10% increments as clinically indicated at five-minute intervals. None of the infants were resuscitated in FiO2421%. On completion of this study, infants were retrospectively divided into subgroups according to the highest grade of
Neonatal blood sampling at 0–24, 24–48, 48–72, 72–96 h and day 7 of life, was paired with routine phlebotomy following fully informed consent from parents. Arterial samples were taken when peripheral or umbilical arterial catheters were in situ, otherwise peripheral venous samples were obtained. At each time point, 1 mL was collected in sodium citrate anticoagulated (whole blood) bottles. Whole blood samples were kept in ice and processed within 90 min and was incubated for one hour in 37 C with pro-inflammatory agent LPS 1 mg/mL to mimic an inflammatory response in vitro [17]. Quantification of respiratory burst activity Generation of ROI was evaluated by flow cytometry using the technique of Smith and Wiedemann [18]. Whole blood (50 mL) was incubated ± LPS (1 mL) at 37 C for 1 h. All samples were subsequently incubated with DHR (100 mM) at 37 C for 10 min before stimulation with 1 mL (16 mM) of PMA for 20 min at 37 C. The reaction was then halted by placing samples on ice. Samples were analyzed using an Accuri C6 flow cytometer with CFlow Plus software (BD Biosciences, Oxford, United Kingdom). Leukocyte populations were selected based on their scatter profiles, forward scatter and side scatter. Neutrophil and monocyte ROI fluorescence intensity was collected on the photomultiplier 2 (FL-2-A) using a 585/40 filter and expressed as mean channel fluorescence. Each sample was acquired over 2 min at medium speed. DHR detects mainly intracellular H2O2 and OH radical production [18]. Quantification of cell surface antigen expression The expression of CD11b and TLR-4 antigens on the surface of neutrophils and monocytes was measured by flow cytometry. Whole blood (50 mL) was treated with 5 mL PE-CD11b and 2.5 mL anti-human TLR-4 antibody and left at 4 C for 20 min. FACS was added and incubated for 10 min at room temperature. The sample was centrifuged at 3000 rpm for five minutes at 4 C. The pellet was suspended twice with DMEM 500 mL and stored on ice before analysis by flow cytometry. Each sample was acquired over two minutes at medium speed, and a minimum of 5000 events were collected and analyzed under the same instrument settings [19].
Neonatal brain injury
DOI: 10.3109/14767058.2014.1000294
Statistics Statistical analysis was carried out using analysis of variance (ANOVA) using PASW statistical package version 18 (Armonk, NY; www.ibm.com/SPSS_Statistics). Two-way ANOVA was used in the comparison of baseline and LPSinduced CD11b and TLR-4 expression between neonates and adults. Equal variance was assumed and Tukey’s post hoc multiple comparisons was used. Chi squared statistic and independent samples t-test were carried out for analysis of demographics. Significance was assumed for values of p50.05. Results are expressed as mean ± standard error of the mean (SEM) unless otherwise indicated.
Results
Downloaded by [University of Otago] at 14:42 27 September 2015
Clinical characteristics There were 122 samples from 22 newborn infants who required resuscitation at delivery (resuscitation only: NE 0: n ¼ 4; NE I: n ¼ 4; NE II: n ¼ 9; and NE III: n ¼ 5). This was a convenience sample taken when FM was available to process the samples. Ten infants were eligible for therapeutic hypothermia, in accordance with the Total Body Hypothermia for Neonatal Encephalopathy criteria [20], and were treated for 72 h duration. Six infants died. There were no differences in gender distribution, birth weight, mode of delivery, Apgar scores, cord pH or admission blood gas parameters between normal and abnormal neuroimaging groups (Table 1). Eighteen infants had a MRI brain in the first week of life with normal imaging (n ¼ 5; score ¼ 0); abnormal signal in the basal ganglia (n ¼ 1; score ¼ 1), abnormal cortical signal (n ¼ 5; score ¼ 2); abnormal signal in both the cortex and basal ganglia/thalamus (n ¼ 2) and widespread abnormal signal involving the entire cortex and basal ganglia/thalamus (n ¼ 5). The remainder had a cranial ultrasound only, which was normal in all cases. CD11b surface expression and NE All neonates, irrespective of insult severity, demonstrated higher basal PMN CD11b expression compared to adults, which was significant in the NE 0/I group on day 3 of life
(p ¼ 0.038) and in NE II/III group at birth, day 4 and day 7 of life (p50.001, p ¼ 0.014 and p ¼ 0.013, respectively; Figure 1a). Following in vitro LPS stimulation, infants with NE had increased surface expression of CD11b to a greater extent than adults. This was significant in both groups at birth (NE 0/I: p ¼ 0.043; NE II/III: p ¼ 0.022) and in the NE II/III group on day 2 of life (p ¼ 0.032; Figure 1b). All NE infants expressed significantly higher monocyte baseline and post LPS stimulation levels of CD11b compared to neonatal controls from birth to 96 h of life. This significantly higher expression level persisted to day 7 of life in the NE II/III group (Figure 1c and d). Higher baseline monocyte CD11b expression was seen in all neonates compared to adults throughout the first week of life, which was statistically significant in NE 0/I neonates on day 3 (p ¼ 0.015) and in NE II/III neonates from birth to day four (0–24 h: p ¼ 0.002; 24–48 h: p ¼ 0.003; 48–72 h: p ¼ 0.007; 72–96 h: p ¼ 0.002; Figure 1c). Neonates with NE II/III displayed significantly elevated post-stimulation CD11b levels compared to adults on day 4 (p ¼ 0.018; Figure 1d). Higher PMN CD11b expression was demonstrated in the abnormal neuroimaging (AI) group and following stimulation with LPS in both neonatal groups at 0–24 h compared to adults. Monocyte CD11b expression was significantly increased at baseline in (a) both neonatal groups from birth to 96 h of life compared to adults and (b) abnormal neuroimaging group compared with neonatal controls on day 7. Following LPS stimulation, CD11b expression was significantly greater tin both neonatal groups compared to neonatal controls from birth to 96 h of life. This persisted in the abnormal neuroimaging group on day 7. Neutrophil and monocyte ROI production is increased in NE All newborns requiring resuscitation at delivery had higher ROI production at baseline in the first 48 h compared to adults and neonatal controls, irrespective of insult severity. Newborns with NE 0/I had comparable ROI levels to adults and neonatal controls thereafter (Figure 2a). Upregulation of ROI production, following LPS stimulation, occurred to a greater magnitude in newborns than in
Table 1. Demographics of infants with neonatal encephalopathy divided by normal versus abnormal neuroimaging.
Male sex (%) GA BW (g) SVD (%) Pre-L Em (%) In-L Em (%) Apgar 1 min Apgar 5 min Cord pH Cord Bxs Admission pH Admission Bxs Admission lactate
3
Normal neuroimaging
Abnormal neuroimaging
p value
64 39+5 (2+0) 3827 (575) 50 43 57 3 (1) 5 (2) 7.05 (0.22) 12.2 (8.4) 7.06 (0.26) 10.4 (11.7) 10.1 (5.7)
56 40+6 (1+0) 3487 (575) 22 0 100 3 (3) 5 (3) 6.97 (0.13) 14.3 (4.0) 7.08 (0.28) 15.3 (10.2) 16.9
0.505 0.133 0.180 0.187 NA NA 0.968 0.738 0.234 0.506 0.860 0.301 0.367
GA, gestational age; BW, birth weight; SVD, spontaneous vaginal delivery; Pre-L Em, prelabour emergency caesarean section; In-L Em, in labour emergency caesarean section; and BE, base excess.
Downloaded by [University of Otago] at 14:42 27 September 2015
4
F. M. O’Hare et al.
J Matern Fetal Neonatal Med, Early Online: 1–8
Figure 1. PMN and monocyte CD11b expression and encephalopathy grade neutrophil and monocyte CD11b expression assessed in healthy adult controls (dark grey), neonatal controls (light grey) (umbilical cord blood), infants requiring resuscitation at birth with no/mild neurological abnormalities (white) (NE 0/I) or moderate/severe abnormality or death (black) (NE II/III) at baseline (a and c) and following LPS stimulation (b and d). *p50.05 versus adult controls, yp50.05 versus neonatal controls. Results expressed as mean channel fluorescence (MCF).
adults or neonatal controls (Figure 2b). Newborns with NE II/ III produced significantly greater ROIs compared to NE 0/I at 72–96 h (p ¼ 0.036) and day 7 of life (p ¼ 0.035; Figure 2b). Monocytes produce 80% less ROIs than neutrophils under the same conditions both at baseline and following stimulation. Neonatal monocytes in both groups produced significantly greater quantities of ROI in the first 48 h of life compared to adult monocytes [NE 0/I: p ¼ 0.001 (0–24 h), p ¼ 0.012 (24–48 h); NE II/III: p50.001, at 0–24 and 24– 48 h, respectively)]. Monocyte ROI production remained significantly higher in infants with NE II/III compared to adults throughout the first week of life. ROI production was higher in NE II/III compared to NE 0/I from birth to day 7 of life and reached statistically significant levels at 48–72 h of life (p ¼ 0.001). All neonates requiring resuscitation at delivery had significantly higher baseline ROI production at birth compared to neonatal controls (NE 0/I: p ¼ 0.012; NE II/ III: p50.001). This elevated ROI production persisted to day 7 of life in the NE II/III group (Figure 2c). Monocyte ROI production was significantly increased following in vitro LPS stimulation in all newborns compared to adults from birth to 48 h of life (NE 0/I: p ¼ 0.019 (0–24 h), p ¼ 0.003 (24–48 h); NE II/III NE: p50.001, at 0–24 and 24–48 h, respectively). Post stimulation, ROI production remained significantly increased in NE II/III compared to adults through to day 7 of life. Both neonatal NE groups had higher post LPS stimulation ROI production compared with neonatal controls.
This reached statistical significance from birth to day 4 of life in the NE II/III group (p50.001 at all time points) and at 24– 48 h in the NE 0/I group (p ¼ 0.015). Greater ROI production was noted in NE II/III monocytes, following LPS stimulation, compared to NE 0/I at 48–72 h (p ¼ 0.002) and 72–96 h of life (p ¼ 0.036; Figure 2d). LPS hyporesponsiveness was initially displayed by the abnormal compared to the normal neuroimaging group with a 5% versus 35% increase in ROI production following LPS stimulation. We also demonstrated an association between ROI production over the first week of life and abnormalities on neuroimaging. Infants requiring resuscitation at delivery with subsequent neuroimaging abnormalities demonstrated increased ROI production compared to infants with normal neuroimaging, especially from day 3 of life onwards (72–96 h; p ¼ 0.060; day 7, p ¼ 0.052; data not shown). LPS hyporesponsiveness was initially found in the abnormal neuroimaging group compared to the normal neuroimaging group (5% versus 35% increase in ROI production following LPS stimulation). Treatment with therapeutic hypothermia (33–34 C for 72 h) had no demonstrable reduction in neutrophil or monocyte ROI production either at baseline or following LPS stimulation. Monocyte ROI production was increased from birth to 48 h of life in all infants requiring resuscitation at delivery, both at baseline and following LPS stimulation, compared to 2 adults. Neutrophil ROI production remained
Neonatal brain injury
Downloaded by [University of Otago] at 14:42 27 September 2015
DOI: 10.3109/14767058.2014.1000294
5
Figure 2. PMN and monocyte ROI production and encephalopathy grade neutrophil and monocyte ROI production assessed in healthy adult controls (dark grey), neonatal controls (light grey) (umbilical cord blood), infants requiring resuscitation at birth with no/mild neurological abnormalities (white) (NE 0/I) or moderate/severe abnormality or death (black) (NE II/III) at baseline (a and c) and following LPS stimulation (b and d)). (a) *p50.05 versus adult controls. (b) *p50.05 versus adult controls, yp50.05 NE 0/I versus NE II/II. (c) *p50.05 versus adult controls, yp50.05 versus neonatal controls. (d) *p50.05 versus adult controls, yp50.05 versus neonatal controls, zp50.05 NE 0/I versus NE II/III. Results expressed as mean channel fluorescence (MCF).
elevated in infants who received therapeutic hypothermia for 72 h compared to adults up to day 7 of life. TLR-4 surface expression and NE There was a significant increase in LPS-stimulated neutrophil TLR-4 in NE non-survivors compared to neonatal controls (p ¼ 0.04). On day 7, there was a significant difference between baseline TLR-4 neonatal controls versus NE survivors (p ¼ 0.031) and stimulated TLR-4 in non-survivors versus survivors (p ¼ 0.044) (data not shown). Baseline PMN TLR-4 expression was significantly increased in both NE groups, compared to adults, at all time points from birth to day 7 of life (Figure 3a). Post LPS stimulation, PMN TLR-4 expression was significantly increased in both NE groups, compared to adults, from birth to day four of life and remained significantly elevated in the NE II/III group on day 7 of life (Figures 3b). Baseline monocyte TLR-4 expression was significantly increased in neonates with NE 0/I compared to adults from birth to 72 h of life and in the NE II/III group from birth to day 7 of life (Figure 3c). All neonates, irrespective of the degree of encephalopathy, expressed higher levels of monocyte TLR-4 following LPS stimulation compared to adults. This was significant in NE II/ III neonates from birth to day 7 of life (0–24 h: p ¼ 0.037; 24– 48 h: p ¼ 0.003; 48–72 h: p ¼ 0.002; 72–96 h: p ¼ 0.001; day 7: p ¼ 0.041; Figure 3d). Basal TLR-4 expression in neonatal
monocytes was significantly elevated at birth compared to adults but not neonatal controls. Therapeutic hypothermia did not significantly alter PMN or monocyte CD11b or TLR-4 expression at any time point during the first week of life.
Discussion We have shown that term neonates following resuscitation at birth have a vigorous systemic innate immune response compared to adults and neonatal controls over the first week of life. Neonates with more severe encephalopathy grades (NE II/III) produced significantly higher basal PMN CD11b levels on days 1, 4 and 7 and monocyte CD11b expression from days 1–4 of life. Neonatal monocyte CD11b expression, irrespective of neuroimaging outcome or encephalopathy grade, was significantly higher than neonatal controls from birth to 96 h of life and remained significantly elevated in the abnormal neuroimaging and NE II/III groups on day 7 of life. Delayed PMN apoptosis with increased CD18/CD11b expression and interleukin (IL)-8 (a PMN chemoattractant) secretion (following LPS stimulation) have been described in neonates suggesting their potential role in neonatal inflammation [21,22]. We have [23] previously reported increased cord PMN CD11b expression in neonates requiring resuscitation at birth with normal neurological outcome and suppression of PMN CD11b expression in neonates with severe neurological signs. Hashimoto et al. [24] found reduced neutrophil
Downloaded by [University of Otago] at 14:42 27 September 2015
6
F. M. O’Hare et al.
J Matern Fetal Neonatal Med, Early Online: 1–8
Figure 3. PMN and monocyte TLR4 expression and encephalopathy grade neutrophil and monocyte TLR-4 expression assessed in healthy adult controls (dark grey), neonatal controls (light grey) (umbilical cord blood), infants requiring resuscitation at birth with no/mild neurological abnormalities (white) (NE 0/I) or moderate/severe abnormality or death (black) (NE II/III) at baseline (a and c) and following LPS stimulation (b and d). *p50.05 versus adult controls. Results expressed as mean channel fluorescence (MCF).
expression of the adhesion molecule L-selectin (shed prior to neutrophil infiltration) in peripheral blood samples from neonates with severe asphyxia within two hours of birth, but did not show any significant differences in PMN CD11b expression between normal neonates and those with mild or severe asphyxia. PMN and monocyte TLR-4 expression was significantly greater than adult levels in the NE II/III group from birth to day 7 of life. Following in vitro LPS stimulation, neonatal PMNs and monocytes upregulated expression to a greater magnitude than adult PMNs. Although TLR-4 expression is increased on adult isolated PMNs under hypoxic conditions, neonatal cord PMNs do not increase TLR-4 expression in vitro [19]. TLR-2 and 4 mRNA is decreased in cord blood mononuclear cells from infants with asphyxia [25]. Animal studies report improved neurological outcomes in TLR-4deficient mice following HI injury and also intracerebral haemorrhage [26]. Increased monocyte TLR-4 correlates with severity of acute cerebral infarction in adults [27]. Specific TLR-4 antagonists decrease brain endothelial activation and neutrophil transmigration in vitro [28]. Persistent inflammation may be associated with persistent activation of hypoxia-inducible factor (HIF) 1 alpha [29]. In addition, upregulated HIF-1a is associated with enhanced antibactericidal activity and phagocytosis and persistent neutrophilic inflammation [30]. In lung ischaemia/reperfusion injury, HIF-1a protein upregulates TLR4 expression in a positive feedback manner [31]. HIF-1a antibody
neutralization attenuates the increase of TLR4 expression in hypoxic microglial cells [32]. TLR4 expression in macrophages is upregulated via HIF-1 in response to hypoxia [33]. Delayed or persistent inflammatory responses have been demonstrated by other groups including a delayed rise in Creactive protein in babies undergoing hypothermia therapy [34]. Youn et al. [35] found persistent elevation of IL-10 and IL-8 in infants with NE and seizures. Vascular endothelial growth factor, glial fibrillary protein and interferon-gamma were persistently elevated in moderate/severe HIE compared to infants with mild HIE at 78–96 h [36]. Advances in neuroimaging techniques have demonstrated that brain injury evolves over days and weeks [37]. Persistence of systemic inflammation was demonstrated as increased systemic ROI production in newborns requiring resuscitation at delivery, which was increased in severe NE. Neutrophil ROI production in adults is decreased in the setting of in vitro and in vivo hypothermia [38]. Therapeutic hypothermia did not significantly alter neutrophil or monocyte ROI production, either at baseline or following LPS stimulation, in our patient population. Other research groups describe a reduction in systemic serum-free radical production in the setting of hypothermia [39] both in vivo and in vitro studies [38], but no other group has studied intracellular ROI production in monocytes and neutrophils, making comparison with other groups difficult. Blocking neutrophil ROI production is a potential target for adjunctive therapy. Many different agents including dexamethasone, IL-10, vitamin A derivatives, vitamin D,
Downloaded by [University of Otago] at 14:42 27 September 2015
DOI: 10.3109/14767058.2014.1000294
vitamin E, uric acid, minocycline and NADPH oxidase inhibition [40] have potential roles as modulators of oxidantmediated injury and decrease ROI production in in vitro studies. This study has several limitations, as comparisons with other studies, which only analysed umbilical cord blood at a single time point, is difficult. Many studies have shown major disparities between cord blood and postnatal neonatal blood inflammatory profiles especially as cord blood is relatively endotoxin tolerant [41]. To our knowledge, there are no human studies to date of TLR-4, CD11b expression and intracellular ROI production in neonatal NE published. Previously Vento et al. have demonstrated increased serum oxidant levels in asphyxiated infants resuscitated with FiO2 100% compared to room air [42]. In this study, we did not measure serum oxidants as several studies have shown a poor correlation with neutrophil intracellular ROIs, and we have shown LPS hyporesponsiveness of neonatal neutrophils compared with adults in hypoxia [43,44]. We were interested in neutrophil and monocyte activation and therefore concentrated on intracellular ROI production. NE remains a significant cause of long-term morbidity and mortality. At present, there are no universally accepted individual markers to pinpoint those asphyxiated infants at highest risk of significant neurological sequelae, although multiple associations have been studied. Further validation of these findings will allow the development of early tests to predict outcome and counsel families. Understanding the immune response in severely affected babies with NE will also allow development of immunomodulatory adjunctive therapies.
Acknowledgements We thank Dr John Murphy, Dr Anne Twomey, Dr Colm O’Donnell and Dr Deirdre Sweetman for all their support and assistance in recruiting patients for this study. We wish to thank all of the parents, babies, laboratory and hospital staff who generously participated in this project. In addition, we wish to thank Dr Bryan Lynch for his expert Neurology opinion and Professor Billy Bourke for his support and encouragement.
Declaration of interest The authors have no further disclosures or conflicts of interest. This study was funded by the National Children’s Research Centre, Crumlin, Dublin 12, Ireland and the National Maternity Hospital Fund, Holles Street, Dublin 2, Ireland.
References 1. Nathan C, Ding A. Non-resolving inflammation. Cell 2010;140: 871–82. 2. Palmer C, Roberts RL, Young PI. Timing of neutrophil depletion influences long-term neuroprotection in neonatal rat hypoxicischemic brain injury. Pediatr Res 2004;55:549–56. 3. Kim JS, Chopp M, Chen H, et al. Adhesive glycoproteins CD11a and CD18 are upregulated in the leukocytes from patients with ischemic stroke and transient ischemic attacks. J Neurol Sci 1995; 128:45–50.
Neonatal brain injury
7
4. Morrison H, McKee D, Ritter L. Systemic neutrophil activation in a mouse model of ischemic stroke and reperfusion. Biol Res Nurs 2011;13:154–63. 5. Matsuo Y, Onodera H, Shiga Y, et al. Role of cell adhesion molecules in brain injury after transient middle cerebral artery occlusion in the rat. Brain Res 1994;656:344–52. 6. Kumar A, Ramakrishna SV, Basu S, Rao GR. Oxidative stress in perinatal asphyxia. Pediatr Neurol 2008;38:181–5. 7. Siesjo BK, Agardh CD, Bengtsson F. Free radicals and brain damage. Cerebrovasc Brain Metab Rev 1989;1:165–211. 8. Chan PH. Role of oxidants in ischemic brain damage. Stroke 1996; 27:1124–9. 9. Jimenez MF, Watson RW, Parodo J, et al. Dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome. Arch Surg 1997;132:1263–9; discussion 1269–70. 10. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5:987–95. 11. Downes CE, Crack PJ. Neural injury following stroke: are Toll-like receptors the link between the immune system and the CNS? Br J Pharmacol 2010;160:1872–88. 12. Tang SC, Arumugam TV, Xu X, et al. Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci USA 2007;104:13798–803. 13. Cao CX, Yang QW, Lv FL, et al. Reduced cerebral ischemiareperfusion injury in Toll-like receptor 4 deficient mice. Biochem Biophys Res Commun 2007;353:509–14. 14. Huang CC, Wang ST, Chang YC, et al. Measurement of the urinary lactate: creatinine ratio for the early identification of newborn infants at risk for hypoxic-ischemic encephalopathy. N Engl J Med 1999;341:328–35. 15. Sarnat HB, Sarnat MS. Neonatal encephalopathy following fetal distress. A clinical and electroencephalographic study. Arch Neurol 1976;33:696–705. 16. Barkovich AJ, Hajnal BL, Vigneron D, et al. Prediction of neuromotor outcome in perinatal asphyxia: evaluation of MR scoring systems. AJNR Am J Neuroradiol 1998;19:143–9. 17. Molloy EJ, O’Neill AJ, Grantham JJ, et al. Labor promotes neonatal neutrophil survival and lipopolysaccharide responsiveness. Pediatr Res 2004;56:99–103. 18. Smith JA, Weidemann MJ. Further characterization of the neutrophil oxidative burst by flow cytometry. J Immunol Methods 1993; 162:261–8. 19. Molloy EJ, O’Neill AJ, Doyle BT, et al. Effects of heat shock and hypoxia on neonatal neutrophil lipopolysaccharide responses: altered apoptosis, Toll-like receptor-4 and CD11b expression compared with adults. Biol Neonate 2006;90:34–9. 20. Azzopardi DV, Strohm B, Edwards AD, et al. Moderate hypothermia to treat perinatal asphyxial encephalopathy. N Engl J Med 2009;361:1349–58. 21. Koenig JM, Stegner JJ, Schmeck AC, et al. Neonatal neutrophils with prolonged survival exhibit enhanced inflammatory and cytotoxic responsiveness. Pediatr Res 2005;57:424–9. 22. Nguyen CN, Schnulle PM, Chegini N, et al. Neonatal neutrophils with prolonged survival secrete mediators associated with chronic inflammation. Neonatology 2010;98:341–7. 23. Molloy EJ, O’Neill AJ, Grantham-Sloan JJ, et al. Neonatal encephalopathy is associated with altered perinatal systemic neutrophil apoptosis. Am J Perinatol 2007;24:525–30. 24. Hashimoto M, Nishida A, Minakami H, et al. Decreased expression of L-selectin on peripheral blood polymorphonuclear leukocytes in neonates with severe asphyxia. Biol Neonate 2002;81: 95–8. 25. Wang L, Xu JB, Tian Y, et al. Expression and significance of Tolllike receptors in cord blood mononuclear cells. Zhonghua Er Ke Za Zhi 2007;45:365–8. 26. Sansing LH, Harris TH, Welsh FA, et al. Toll-like receptor 4 contributes to poor outcome after intracerebral hemorrhage. Ann Neurol 2011;70:646–56. 27. Yang QW, Li JC, Lu FL, et al. Upregulated expression of toll-like receptor 4 in monocytes correlates with severity of acute cerebral infarction. J Cereb Blood Flow Metab 2008;28:1588–96. 28. Leow-Dyke S, Allen C, Denes A, et al. Neuronal toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro. J Neuroinflammation 2012;9: 230.
Downloaded by [University of Otago] at 14:42 27 September 2015
8
F. M. O’Hare et al.
29. Anand RJ, Gribar SC, Li J, et al. Hypoxia causes an increase in phagocytosis by macrophages in a HIF-1alpha-dependent manner. J Leukoc Biol 2007;82:1257–65. 30. McGovern NN, Cowburn AS, Porter L, et al. Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils. J Immunol 2011;186:453–63. 31. Zhou Z, Zhu X, Chen J, et al. The interaction between Tolllike receptor 4 signaling pathway and hypoxia-inducible factor 1a in lung ischemia-reperfusion injury. J Surg Res 2014;188: 290–7. 32. Yao L, Kan EM, Lu J, et al. Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia. J Neuroinflammation 2013;10:23. doi:10.1186/1742-2094-10-23. 33. Kim SY, Choi YJ, Joung SM, et al. Hypoxic stress up-regulates the expression of Toll-like receptor 4 in macrophages via hypoxiainducible factor. Immunology 2010;129:516–24. 34. Chakkarapani E, Davis J, Thoresen M. Therapeutic hypothermia delays the C-reactive protein response and suppresses white blood cell and platelet count in infants with neonatal encephalopathy. Arch Dis Child Fetal Neonatal Ed 2014;99:F458–63. 35. Youn YA, Kim SJ, Sung IK, et al. Serial examination of serum IL-8, IL-10 and IL-1Ra levels is significant in neonatal seizures induced by hypoxic-ischaemic encephalopathy. Scand J Immunol 2012;76:286–93. 36. Chalak LF, Sa´nchez PJ, Adams-Huet B, et al. Biomarkers for severity of neonatal hypoxic-ischemic encephalopathy and
J Matern Fetal Neonatal Med, Early Online: 1–8
37. 38. 39. 40. 41.
42. 43.
44.
outcomes in newborns receiving hypothermia therapy. J Pediatr 2014;164:468–74. McKinstry RC, Miller JH, Snyder AZ, et al. A prospective, longitudinal diffusion tensor imaging study of brain injury in newborns. Neurology 2002;59:824–33. Wenisch C, Narzt E, Sessler DI, et al. Mild intraoperative hypothermia reduces production of reactive oxygen intermediates by polymorphonuclear leukocytes. Anesth Analg 1996;82:810–16. Globus MY, Alonso O, Dietrich WD, et al. Glutamate release and free radical production following brain injury: effects of posttraumatic hypothermia. J Neurochem 1995;65:1704–11. Chen H, Kim GS, Okami N, et al. NADPH oxidase is involved in post-ischemic brain inflammation. Neurobiol Dis 2011;42:341–8. Gessler P, Neu S, Nebe T, Speer CP. Granulocyte colonystimulating factor receptor expression on neutrophils of term and preterm neonates with and without signs of infection. Eur J Pediatr 1999;158:497–500. Vento M, Asensi M, Sastre J, et al. Oxidative stress in asphyxiated term infants resuscitated with 100% oxygen. J Pediatr 2003;142: 240–6. Maor I, Rainis T, Lanir A, Lavy A. Oxidative stress, inflammation and neutrophil superoxide release in patients with Crohn’s disease: distinction between active and non-active disease. Dig Dis Sci 2008;53:2208–14. Molloy EJ, O’Neill AJ, Doyle BT, et al. Effects of heat shock and hypoxia on neonatal neutrophil lipopolysaccharide responses: altered apoptosis, Toll-like receptor-4 and CD11b expression compared with adults. Biol Neonate 2006;90:34–9.
