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

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 (  [  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  <  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 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

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

Serial blood cytokine and chemokine mRNA and microRNA over 48 h are insult specific in a piglet model of inflammation-sensitized hypoxia-ischaemia.

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 < 0.01) at 1-3 h and ENO2 (R = -0.69; p = 0.01) at 48 h. CONCLUSIONS: mRNA and miRNA differentiated hypoxia from inflammation-sensitized hypoxia within 6 h in a piglet model. This information may inform human studies to enable triage for tailored neuroprotection in NE. IMPACT: Early stratification of infants with neonatal encephalopathy is key to providing tailored neuroprotection. IL1A, CXCL8, IL10, CCL2 and NSE mRNA are promising biomarkers of inflammation-sensitized hypoxia. IL10 mRNA levels differentiated all three pathological states; fold changes from baseline was the highest in LPS+Hypoxia animals, followed by LPS and Hypoxia at 6 h. miR-23, -27, -31-5p and -193-5p were significantly upregulated within 6 h of a hypoxia insult. Functional analysis highlighted the diverse roles of miRNA in cellular processes

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

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 ( 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  Conclusions Our findings support a potential role for IL-10 as a biomarker for adverse outcomes after neonatal encephalopathy.</p