Elevated serum IL-10 is associated with severity of neonatal encephalopathy and adverse early childhood outcomes.

BACKGROUND: Neonatal encephalopathy (NE) contributes substantially to child mortality and disability globally. We compared cytokine profiles in term Ugandan neonates with and without NE, with and without perinatal infection or inflammation and identified biomarkers predicting neonatal and early childhood outcomes. METHODS: In this exploratory biomarker study, serum IL-1α, IL-6, IL-8, IL-10, TNFα, and VEGF (<12 h) were compared between NE and non-NE infants with and without perinatal infection/inflammation. Neonatal (severity of NE, mortality) and early childhood (death or neurodevelopmental impairment to 2.5 years) outcomes were assessed. Predictors of outcomes were explored with multivariable linear and logistic regression and receiver-operating characteristic analyses. RESULTS: Cytokine assays on 159 NE and 157 non-NE infants were performed; data on early childhood outcomes were available for 150 and 129, respectively. NE infants had higher IL-10 (p < 0.001), higher IL-6 (p < 0.017), and lower VEGF (p < 0.001) levels. Moderate and severe NE was associated with higher IL-10 levels compared to non-NE infants (p < 0.001). Elevated IL-1α was associated with perinatal infection/inflammation (p = 0.013). Among NE infants, IL-10 predicted neonatal mortality (p = 0.01) and adverse early childhood outcome (adjusted OR 2.28, 95% CI 1.35-3.86, p = 0.002). CONCLUSIONS: Our findings support a potential role for IL-10 as a biomarker for adverse outcomes after neonatal encephalopathy. IMPACT: Neonatal encephalopathy is a common cause of child death and disability globally. Inflammatory cytokines are potential biomarkers of encephalopathy severity and outcome. In this Ugandan health facility-based cohort, neonatal encephalopathy was associated with elevated serum IL-10 and IL-6, and reduced VEGF at birth. Elevated serum IL-10 within 12 h after birth predicted severity of neonatal encephalopathy, neonatal mortality, and adverse early childhood developmental outcomes, independent of perinatal infection or inflammation, and provides evidence to the contribution of the inflammatory processes. Our findings support a role for IL-10 as a biomarker for adverse outcomes after neonatal encephalopathy in a sub-Saharan African cohort.

Hypothermia is not therapeutic in a neonatal piglet model of inflammation-sensitized hypoxia-ischemia.

BACKGROUND: Perinatal inflammation combined with hypoxia-ischemia (HI) exacerbates injury in the developing brain. Therapeutic hypothermia (HT) is standard care for neonatal encephalopathy; however, its benefit in inflammation-sensitized HI (IS-HI) is unknown. METHODS: Twelve newborn piglets received a 2 µg/kg bolus and 1 µg/kg/h infusion over 52 h of Escherichia coli lipopolysaccharide (LPS). HI was induced 4 h after LPS bolus. After HI, piglets were randomized to HT (33.5 °C 1-25 h after HI, n = 6) or normothermia (NT, n = 6). Amplitude-integrated electroencephalogram (aEEG) was recorded and magnetic resonance spectroscopy (MRS) was acquired at 24 and 48 h. At 48 h, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive brain cell death, microglial activation/proliferation, astrogliosis, and cleaved caspase-3 (CC3) were quantified. Hematology and plasma cytokines were serially measured. RESULTS: Two HT piglets died. aEEG recovery, thalamic and white matter MRS lactate/N-acetylaspartate, and TUNEL-positive cell death were similar between groups. HT increased microglial activation in the caudate, but had no other effect on glial activation/proliferation. HT reduced CC3 overall. HT suppressed platelet count and attenuated leukocytosis. Cytokine profile was unchanged by HT. CONCLUSIONS: We did not observe protection with HT in this piglet IS-HI model based on aEEG, MRS, and immunohistochemistry. Immunosuppressive effects of HT and countering neuroinflammation by LPS may contribute to the observed lack of HT efficacy. Other immunomodulatory strategies may be more effective in IS-HI. IMPACT: Acute infection/inflammation is known to exacerbate perinatal brain injury and can worsen the outcomes in neonatal encephalopathy. Therapeutic HT is the current standard of care for all infants with NE, but the benefit in infants with coinfection/inflammation is unknown. In a piglet model of inflammation (LPS)-sensitized HI, we observed no evidence of neuroprotection with cooling for 24 h, based on our primary outcome measures: aEEG, MRS Lac/NAA, and histological brain cell death. Additional neuroprotective agents, with beneficial immunomodulatory effects, require exploration in IS-HI models.

Human umbilical cord mesenchymal stromal cells as an adjunct therapy with therapeutic hypothermia in a piglet model of perinatal asphyxia.

BACKGROUND: With therapeutic hypothermia (HT) for neonatal encephalopathy, disability rates are reduced, but not all babies benefit. Pre-clinical rodent studies suggest mesenchymal stromal cells (MSCs) augment HT protection. AIMS: The authors studied the efficacy of intravenous (IV) or intranasal (IN) human umbilical cord-derived MSCs (huMSCs) as adjunct therapy to HT in a piglet model. METHODS: A total of 17 newborn piglets underwent transient cerebral hypoxia-ischemia (HI) and were then randomized to (i) HT at 33.5°C 1-13 h after HI (n = 7), (ii) HT+IV huMSCs (30 × 106 cells) at 24 h and 48 h after HI (n = 5) or (iii) HT+IN huMSCs (30 × 106 cells) at 24 h and 48 h after HI (n = 5). Phosphorus-31 and hydrogen-1 magnetic resonance spectroscopy (MRS) was performed at 30 h and 72 h and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and oligodendrocytes quantified. In two further piglets, 30 × 106 IN PKH-labeled huMSCs were administered. RESULTS: HI severity was similar between groups. Amplitude-integrated electroencephalogram (aEEG) recovery was more rapid for HT+IN huMSCs compared with HT from 25 h to 42 h and 49 h to 54 h (P ≤ 0.05). MRS phosphocreatine/inorganic phosphate was higher on day 2 in HT+IN huMSCs than HT (P = 0.035). Comparing HT+IN huMSCs with HT and HT+IV huMSCs, there were increased OLIG2 counts in hippocampus (P = 0.011 and 0.018, respectively), internal capsule (P = 0.013 and 0.037, respectively) and periventricular white matter (P = 0.15 for IN versus IV huMSCs). Reduced TUNEL-positive cells were seen in internal capsule with HT+IN huMSCs versus HT (P = 0.05). PKH-labeled huMSCs were detected in the brain 12 h after IN administration. CONCLUSIONS: After global HI, compared with HT alone, the authors saw beneficial effects of HT+IN huMSCs administered at 24 h and 48 h (30 × 106 cells/kg total dose) based on more rapid aEEG recovery, improved 31P MRS brain energy metabolism and increased oligodendrocyte survival at 72 h.

Proton Magnetic Resonance Spectroscopy Lactate/N-Acetylaspartate Within 48 h Predicts Cell Death Following Varied Neuroprotective Interventions in a Piglet Model of Hypoxia-Ischemia With and Without Inflammation-Sensitization.

Despite therapeutic hypothermia, survivors of neonatal encephalopathy have high rates of adverse outcome. Early surrogate outcome measures are needed to speed up the translation of neuroprotection trials. Thalamic lactate (Lac)/N-acetylaspartate (NAA) peak area ratio acquired with proton (1H) magnetic resonance spectroscopy (MRS) accurately predicts 2-year neurodevelopmental outcome. We assessed the relationship between MR biomarkers acquired at 24-48 h following injury with cell death and neuroinflammation in a piglet model following various neuroprotective interventions. Sixty-seven piglets with hypoxia-ischemia, hypoxia alone, or lipopolysaccharide (LPS) sensitization were included, and neuroprotective interventions were therapeutic hypothermia, melatonin, and magnesium. MRS and diffusion-weighted imaging (DWI) were acquired at 24 and 48 h. At 48 h, experiments were terminated, and immunohistochemistry was assessed. There was a correlation between Lac/NAA and overall cell death [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)] [mean Lac/NAA basal ganglia and thalamus (BGT) voxel r = 0.722, white matter (WM) voxel r = 0.784, p < 0.01] and microglial activation [ionized calcium-binding adapter molecule 1 (Iba1)] (BGT r = -0.786, WM r = -0.632, p < 0.01). Correlation with marker of caspase-dependent apoptosis [cleaved caspase 3 (CC3)] was lower (BGT r = -0.636, WM r = -0.495, p < 0.01). Relation between DWI and TUNEL was less robust (mean diffusivity BGT r = -0.615, fractional anisotropy BGT r = 0.523). Overall, Lac/NAA correlated best with cell death and microglial activation. These data align with clinical studies demonstrating Lac/NAA superiority as an outcome predictor in neonatal encephalopathy (NE) and support its use in preclinical and clinical neuroprotection studies.

High-Dose Melatonin and Ethanol Excipient Combined with Therapeutic Hypothermia in a Newborn Piglet Asphyxia Model.

With the current practice of therapeutic hypothermia for neonatal encephalopathy, disability rates and the severity spectrum of cerebral palsy are reduced. Nevertheless, safe and effective adjunct therapies are needed to optimize outcomes. This study's objective was to assess if 18 mg/kg melatonin given rapidly over 2 h at 1 h after hypoxia-ischemia with cooling from 1-13 h was safe, achieved therapeutic levels within 3 h and augmented hypothermic neuroprotection. Following hypoxia-ischemia, 20 newborn piglets were randomized to: (i) Cooling 1-13 h (HT; n = 6); (ii) HT+ 2.5% ethanol vehicle (HT+V; n = 7); (iii) HT + Melatonin (HT+M; n = 7). Intensive care was maintained for 48 h; aEEG was acquired throughout, brain MRS acquired at 24 and 48 h and cell death (TUNEL) evaluated at 48 h. There were no differences for insult severity. Core temperature was higher in HT group for first hour after HI. Comparing HT+M to HT, aEEG scores recovered more quickly by 19 h (p < 0.05); comparing HT+V to HT, aEEG recovered from 31 h (p < 0.05). Brain phosphocreatine/inorganic phosphate and NTP/exchangeable phosphate were higher at 48 h in HT+M versus HT (p = 0.036, p = 0.049 respectively). Including both 24 h and 48 h measurements, the rise in Lactate/N-acetyl aspartate was reduced in white (p = 0.030) and grey matter (p = 0.038) after HI. Reduced overall TUNEL positive cells were observed in HT+M (47.1 cells/mm2) compared to HT (123.8 cells/mm2) (p = 0.0003) and HT+V (97.5 cells/mm2) compared to HT (p = 0.012). Localized protection was seen in white matter for HT+M versus HT (p = 0.036) and internal capsule for HT+M compared to HT (p = 0.001) and HT+V versus HT (p = 0.006). Therapeutic melatonin levels (15-30mg/l) were achieved at 2 h and were neuroprotective following HI, but ethanol vehicle was partially protective.

Quantification of the severity of hypoxic-ischemic brain injury in a neonatal preclinical model using measurements of cytochrome-c-oxidase from a miniature broadband-near-infrared spectroscopy system.

We describe the development of a miniaturized broadband near-infrared spectroscopy system (bNIRS), which measures changes in cerebral tissue oxyhemoglobin ( [ HbO 2 ] ) and deoxyhemoglobin ([HHb]) plus tissue metabolism via changes in the oxidation state of cytochrome-c-oxidase ([oxCCO]). The system is based on a small light source and a customized mini-spectrometer. We assessed the instrument in a preclinical study in 27 newborn piglets undergoing transient cerebral hypoxia-ischemia (HI). We aimed to quantify the recovery of the HI insult and estimate the severity of the injury. The recovery in brain oxygenation ( Δ [ HbDiff ] = Δ [ HbO 2 ] - Δ [ HHb ] ), blood volume ( Δ [ HbT ] = Δ [ HbO 2 ] + Δ [ HHb ] ), and metabolism ( Δ [ oxCCO ] ) for up to 30 min after the end of HI were quantified in percentages using the recovery fraction (RF) algorithm, which quantifies the recovery of a signal with respect to baseline. The receiver operating characteristic analysis was performed on bNIRS-RF measurements compared to proton ( H 1 ) magnetic resonance spectroscopic (MRS)-derived thalamic lactate/N-acetylaspartate (Lac/NAA) measured at 24-h post HI insult; Lac/NAA peak area ratio is an accurate surrogate marker of neurodevelopmental outcome in babies with neonatal HI encephalopathy. The Δ [ oxCCO ] -RF cut-off threshold of 79% within 30 min of HI predicted injury severity based on Lac/NAA with high sensitivity (100%) and specificity (93%). A significant difference in thalamic Lac/NAA was noticed ( p < 0.0001 ) between the two groups based on this cut-off threshold of 79% Δ [ oxCCO ] -RF. The severe injury group ( n = 13 ) had ∼ 30 % smaller recovery in Δ [ HbDiff ] -RF ( p = 0.0001 ) and no significant difference was observed in Δ [ HbT ] -RF between groups. At 48 h post HI, significantly higher P 31 -MRS-measured inorganic phosphate/exchangeable phosphate pool (epp) ( p = 0.01 ) and reduced phosphocreatine/epp ( p = 0.003 ) were observed in the severe injury group indicating persistent cerebral energy depletion. Based on these results, the bNIRS measurement of the oxCCO recovery fraction offers a noninvasive real-time biomarker of brain injury severity within 30 min following HI insult.

Acute LPS sensitization and continuous infusion exacerbates hypoxic brain injury in a piglet model of neonatal encephalopathy.

Co-existing infection/inflammation and birth asphyxia potentiate the risk of developing neonatal encephalopathy (NE) and adverse outcome. In a newborn piglet model we assessed the effect of E. coli lipopolysaccharide (LPS) infusion started 4 h prior to and continued for 48 h after hypoxia on brain cell death and systemic haematological changes compared to LPS and hypoxia alone. LPS sensitized hypoxia resulted in an increase in mortality and in brain cell death (TUNEL positive cells) throughout the whole brain, and in the internal capsule, periventricular white matter and sensorimotor cortex. LPS alone did not increase brain cell death at 48 h, despite evidence of neuroinflammation, including the greatest increases in microglial proliferation, reactive astrocytosis and cleavage of caspase-3. LPS exposure caused splenic hypertrophy and platelet count suppression. The combination of LPS and hypoxia resulted in the highest and most sustained systemic white cell count increase. These findings highlight the significant contribution of acute inflammation sensitization prior to an asphyxial insult on NE illness severity.

Core temperature after birth in babies with neonatal encephalopathy in a sub-Saharan African hospital setting.

KEY POINTS: Therapeutic hypothermia (HT) to 33.0-34.0°C for 72 h provides optimal therapy for infants with neonatal encephalopathy (NE) in high-resource settings. HT is not universally implemented in low- and middle-income countries as a result of both limited resources and evidence. Facilitated passive cooling, comprising infants being allowed to passively lower their body temperature in the days after birth, is an emerging practice in some West African neonatal units. In this observational study, we demonstrate that infants undergoing facilitated passive cooling in a neonatal unit in Accra, Ghana, achieve temperatures within the HT target range ∼20% of the 72 h. Depth of HT fluctuates and can be excessive, as well as not maintained, especially after 24 h. Sustained and deeper passive cooling was evident for severe NE and for those that died. It is important to prevent excessive cooling, to understand that severe NE babies cool more and to be aware of facilitated passive cooling with respect to the design of clinical trials in low- and mid-resource settings. ABSTRACT: Neonatal encephalopathy (NE) is a significant worldwide problem with the greatest burden in sub-Saharan Africa. Therapeutic hypothermia (HT), comprising the standard of care for infants with moderate-to-severe NE in settings with sophisticated intensive care, is not available to infants in many sub-Saharan African countries, including Ghana. We prospectively assessed the temperature response in relation to outcome in the 80 h after birth in a cohort of babies with NE undergoing 'facilitated passive cooling' at Korle Bu Teaching Hospital, Accra, Ghana. We hypothesized that NE infants demonstrate passive cooling. Thirteen infants (69% male) ≥36 weeks with moderate-to-severe NE were enrolled. Ambient mean ± SD temperature was 28.3 ± 0.7°C. Infant core temperature was 34.2 ± 1.2°C over the first 24 h and 35.0 ± 1.0°C over 80 h. Nadir mean temperature occurred at 15 h. Temperatures were within target range for HT with respect to 18 ± 14% of measurements within the first 72 h. Axillary temperature was 0.5 ± 0.2°C below core. Three infants died before discharge. Core temperature over 80 h for surviving infants was 35.3 ± 0.9°C and 33.96 ± 0.7°C for those that died (P = 0.043). Temperature profile negatively correlated with Thompson NE score on day 4 (r2  = 0.66): infants with a Thompson score of 0-6 had higher temperatures than those with a score of 7-15 (P = 0.021) and a score of 16+/deceased (P = 0.007). More severe NE was associated with lower core temperatures. Passive cooling is a physiological response after hypoxia-ischaemia; however, the potential neuroprotective effect of facilitated passive cooling is unknown. An awareness of facilitated passive cooling in babies with NE is important for the design of clinical trials of neuroprotection in low and mid resource settings.

Neonatal Encephalopathy With Group B Streptococcal Disease Worldwide: Systematic Review, Investigator Group Datasets, and Meta-analysis.

BACKGROUND: Neonatal encephalopathy (NE) is a leading cause of child mortality and longer-term impairment. Infection can sensitize the newborn brain to injury; however, the role of group B streptococcal (GBS) disease has not been reviewed. This paper is the ninth in an 11-article series estimating the burden of GBS disease; here we aim to assess the proportion of GBS in NE cases. METHODS: We conducted systematic literature reviews (PubMed/Medline, Embase, Latin American and Caribbean Health Sciences Literature [LILACS], World Health Organization Library Information System [WHOLIS], and Scopus) and sought unpublished data from investigator groups reporting GBS-associated NE. Meta-analyses estimated the proportion of GBS disease in NE and mortality risk. UK population-level data estimated the incidence of GBS-associated NE. RESULTS: Four published and 25 unpublished datasets were identified from 13 countries (N = 10436). The proportion of NE associated with GBS was 0.58% (95% confidence interval [CI], 0.18%-.98%). Mortality was significantly increased in GBS-associated NE vs NE alone (risk ratio, 2.07 [95% CI, 1.47-2.91]). This equates to a UK incidence of GBS-associated NE of 0.019 per 1000 live births. CONCLUSIONS: The consistent increased proportion of GBS disease in NE and significant increased risk of mortality provides evidence that GBS infection contributes to NE. Increased information regarding this and other organisms is important to inform interventions, especially in low- and middle-resource contexts.