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BACKGROUND: Exposure to inflammation exacerbates injury in neonatal encephalopathy (NE). We hypothesized that brain biomarker mRNA, cytokine mRNA and microRNA differentiate inflammation (E. coli LPS), hypoxia (Hypoxia), and inflammation-sensitized hypoxia (LPS+Hypoxia) in an NE piglet model. METHODS: Sixteen piglets were randomized: (i) LPS 2 μg/kg bolus; 1 μg/kg infusion (LPS; n = 5), (ii) Saline with hypoxia (Hypoxia; n = 6), (iii) LPS commencing 4 h pre-hypoxia (LPS+Hypoxia; n = 5). Total RNA was acquired at baseline, 4 h after LPS and 1, 3, 6, 12, 24, 48 h post-insult (animals euthanized at 48 h). Quantitative PCR was performed for cytokines (IL1A, IL6, CXCL8, IL10, TNFA) and brain biomarkers (ENO2, UCHL1, S100B, GFAP, CRP, BDNF, MAPT). MicroRNA was detected using GeneChip (Affymetrix) microarrays. Fold changes from baseline were compared between groups and correlated with cell death (TUNEL) at 48 h. RESULTS: Within 6 h post-insult, we observed increased IL1A, CXCL8, CCL2 and ENO2 mRNA in LPS+Hypoxia and LPS compared to Hypoxia. IL10 mRNA differentiated all groups. Four microRNAs differentiated LPS+Hypoxia and Hypoxia: hsa-miR-23a, 27a, 31-5p, 193-5p. Cell death correlated with TNFA (R = 0.69; p

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.

Neonatal encephalopathy is a leading cause of morbidity and mortality worldwide. Although therapeutic hypothermia (HT) is now standard practice in most neonatal intensive care units in high resource settings, some infants still develop long-term adverse neurological sequelae. In low resource settings, HT may not be safe or efficacious. Therefore, additional neuroprotective interventions are urgently needed. Melatonin’s diverse neuroprotective properties include antioxidant, anti-inflammatory, and anti-apoptotic effects. Its strong safety profile and compelling preclinical data suggests that melatonin is a promising agent to improve the outcomes of infants with NE. Over the past decade, the safety and efficacy of melatonin to augment HT has been studied in the neonatal piglet model of perinatal asphyxia. From this model, we have observed that the neuroprotective effects of melatonin are time-critical and dose dependent. Therapeutic melatonin levels are likely to be 15–30 mg/L and for optimal effect, these need to be achieved within the first 2–3 h after birth. This review summarises the neuroprotective properties of melatonin, the key findings from the piglet and other animal studies to date, and the challenges we face to translate melatonin from bench to bedside.

Hypoxic ischaemic encephalopathy (HIE) is a significant cause of death and disability. Therapeutic hypothermia (TH) is the only available standard of treatment, but 45–55% of cases still result in death or neurodevelopmental disability following TH. This work has focussed on developing a new brain tissue physiology and biochemistry systems biology model that includes temperature effects, as well as a Bayesian framework for analysis of model parameter estimation. Through this, we can simulate the effects of temperature on brain tissue oxygen delivery and metabolism, as well as analyse clinical and experimental data to identify mechanisms to explain differing behaviour and outcome. Presented here is an application of the model to data from two piglets treated with TH following hypoxic-ischaemic injury showing different responses and outcome following treatment. We identify the main mechanism for this difference as the Q10 temperature coefficient for metabolic reactions, with the severely injured piglet having a median posterior value of 0.133 as opposed to the mild injury value of 5.48. This work demonstrates the use of systems biology models to investigate underlying mechanisms behind the varying response to hypothermic treatment.

As therapeutic hypothermia is only partially protective for neonatal encephalopathy, safe and effective adjunct therapies are urgently needed. Melatonin and erythropoietin show promise as safe and effective neuroprotective therapies. We hypothesized that melatonin and erythropoietin individually augment 12-h hypothermia (double therapies) and hypothermia + melatonin + erythropoietin (triple therapy) leads to optimal brain protection. Following carotid artery occlusion and hypoxia, 49 male piglets (, As therapeutic hypothermia is partially protective in neonatal encephalopathy, adjunct therapies are required. In a piglet model of perinatal asphyxia, Pang et al. report that melatonin and erythropoietin are safe and individually augment cooling through different mechanisms. Staggered rather than concomitant dosing may be optimal for brain protection., Graphical Abstract Graphical Abstract

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.

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 degrees C 1-13 h after HI (n = 7), (ii) HT+IV huMSCs (30 x 10(6) cells) at 24 h and 48 h after HI (n = 5) or (iii) HT+IN huMSCs (30 x 10(6) 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 x 10(6) 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

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 2) 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.

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.

Therapeutic hypothermia is only partially protective for neonatal encephalopathy; there is an urgent need to develop treatments that augment cooling. Our objective was to assess safety, efficacy and pharmacokinetics of 5 and 15 mg/kg/24 h melatonin (proprietary formulation) administered at 2 h and 26 h after hypoxia-ischemia (HI) with cooling in a piglet model. Following moderate cerebral HI, 30 piglets were eligible and randomized to: i) Hypothermia (33.5 °C, 2–26 h) and vehicle (HT + V;n = 13); b) HT and 5 mg/kg melatonin over 6 h at 2 h and 26 h after HI (HT + Mel-5;n = 4); c) HT and 15 mg/kg melatonin over 6 h at 2 h and 26 h after HI (HT + Mel-15;n = 13). Intensive care was maintained for 48 h; brain MRS was acquired and cell death (TUNEL) evaluated at 48 h. Comparing HT + V with HT + Mel-5 and HT + Mel-15, there was no difference in blood pressure or inotropic support needed, brain Lactate/N Acetylaspartate at 24 h and 48 h was similar, ATP/phosphate pool was higher for HT + Mel-15 versus HT + V at 24 h (p = 0.038) but not 48 h. A localized reduction in TUNEL positive cell death was observed in the sensorimotor cortex in the 15 mg/kg melatonin group (HT + Mel-15 versus HT + V; p

We describe the development of a miniaturized broadband near-infrared spectroscopy system (bNIRS), which measures changes in cerebral tissue oxyhemoglobin ( [ HbO2 ] ) 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 [ HbO2 ] − Δ [ HHb ] ), blood volume (Δ [ HbT ] = Δ [ HbO2 ] + 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 (H1) 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 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 P31-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.

This paper argues that there is a need to make a distinction between 'spiritual development' (educational in intent, relevant for all) and 'developing spirituality' (catechetical in intent and therefore inappropriate for some). Making this distinction resolves much of the confusion in the spiritual development debate that results from (i) problems concerning definition; (ii) co-terminous usage of related but distinct concepts; and (iii) a plurality of competing models of spiritual development. This confusion is illustrated with reference to a variety of authors, particularly Hay, Erricker, Wright, Bradford, Mott-Thornton, Carr and Thatcher. Further, distinguishing between 'spiritual development' and 'developing spirituality' will bring clearer thinking to discussions about spiritual education and will overcome some of the weaknesses in the work of specified authors.

This paper argues that a twin‐track approach to spiritual provision that distinguishes ‘spiritual development’ (educational in intent, relevant for all) from ‘developing spirituality’ (catechetical in intent and therefore inappropriate for some) enables Catholic sixth form colleges to fulfil both educational and catechetical aims, as appropriate. Distinguishing between ‘spiritual development’ and ‘developing spirituality’ allows Catholic colleges to take spiritual education seriously whilst simultaneously fulfilling the aims of Catholic education to (1) provide for catechesis; (2) promote dialogue with other faith traditions and with secular society; and (3) provide appropriate holistic education for all students. Construing ‘spiritual development’ in exclusive religious terms disallows Catholic colleges, with their plural intakes, from providing spiritual development for all. In other words, the distinction proposed here a/lows Catholic colleges to be genuinely Catholic and at the same time to engage in ...

This paper asserts that the spiritual is rooted in the essence of being human. Human beings search for meaning, purpose and direction in life and spiritual development is inextricably linked with this process. The spiritual manifests itself in the feelings and emotions of human beings, as well as the values, beliefs, aspirations and intellect. Spiritual development in this sense must be holistic. The spiritual dimension of education must engage the body, mind and heart. Spiritual development implies change, with a progression from immaturity towards maturity, from the simple to the complex, from the superficial to the profound. In relation to this the writer discusses what spiritual education should involve and the contribution different aspects of the curriculum can make.