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.

Melatonin and/or erythropoietin combined with hypothermia in a piglet model of perinatal asphyxia.

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 (<48 h old) were randomized to: (i) hypothermia + vehicle (n = 12), (ii) hypothermia + melatonin (20 mg/kg over 2 h) (n = 12), (iii) hypothermia + erythropoietin (3000 U/kg bolus) (n = 13) or (iv) triple therapy (n = 12). Melatonin, erythropoietin or vehicle were given at 1, 24 and 48 h after hypoxia-ischaemia. Hypoxia-ischaemia severity was similar across groups. Therapeutic levels were achieved 3 hours after hypoxia-ischaemia for melatonin (15-30 mg/l) and within 30 min of erythropoietin administration (maximum concentration 10 000 mU/ml). Compared to hypothermia + vehicle, we observed faster amplitude-integrated EEG recovery from 25 to 30 h with hypothermia + melatonin (P = 0.02) and hypothermia + erythropoietin (P = 0.033) and from 55 to 60 h with triple therapy (P = 0.042). Magnetic resonance spectroscopy lactate/N-acetyl aspartate peak ratio was lower at 66 h in hypothermia + melatonin (P = 0.012) and triple therapy (P = 0.032). With hypothermia + melatonin, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelled-positive cells were reduced in sensorimotor cortex (P = 0.017) and oligodendrocyte transcription factor 2 labelled-positive counts increased in hippocampus (P = 0.014) and periventricular white matter (P = 0.039). There was no reduction in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelled-positive cells with hypothermia + erythropoietin, but increased oligodendrocyte transcription factor 2 labelled-positive cells in 5 of 8 brain regions (P < 0.05). Overall, melatonin and erythropoietin were safe and effective adjunct therapies to hypothermia. Hypothermia + melatonin double therapy led to faster amplitude-integrated EEG recovery, amelioration of lactate/N-acetyl aspartate rise and reduction in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelled-positive cells in the sensorimotor cortex. Hypothermia + erythropoietin double therapy was in association with EEG recovery and was most effective in promoting oligodendrocyte survival. Triple therapy provided no added benefit over the double therapies in this 72-h study. Melatonin and erythropoietin influenced cell death and oligodendrocyte survival differently, reflecting distinct neuroprotective mechanisms which may become more visible with longer-term studies. Staggering the administration of therapies with early melatonin and later erythropoietin (after hypothermia) may provide better protection; each therapy has complementary actions which may be time critical during the neurotoxic cascade after hypoxia-ischaemia.

Nutritional Supplementation Reduces Lesion Size and Neuroinflammation in a Sex-Dependent Manner in a Mouse Model of Perinatal Hypoxic-Ischemic Brain Injury.

Perinatal hypoxia-ischemia (HI) is a major cause of neonatal brain injury, leading to long-term neurological impairments. Medical nutrition can be rapidly implemented in the clinic, making it a viable intervention to improve neurodevelopment after injury. The omega-3 (n-3) fatty acids docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), uridine monophosphate (UMP) and choline have previously been shown in rodents to synergistically enhance brain phospholipids, synaptic components and cognitive performance. The objective of this study was to test the efficacy of an experimental diet containing DHA, EPA, UMP, choline, iodide, zinc, and vitamin B12 in a mouse model of perinatal HI. Male and female C57Bl/6 mice received the experimental diet or an isocaloric control diet from birth. Hypoxic ischemic encephalopathy was induced on postnatal day 9 by ligation of the right common carotid artery and systemic hypoxia. To assess the effects of the experimental diet on long-term motor and cognitive outcome, mice were subjected to a behavioral test battery. Lesion size, neuroinflammation, brain fatty acids and phospholipids were analyzed at 15 weeks after HI. The experimental diet reduced lesion size and neuroinflammation specifically in males. In both sexes, brain n-3 fatty acids were increased after receiving the experimental diet. The experimental diet also improved novel object recognition, but no significant effects on motor performance were observed. Current data indicates that early life nutritional supplementation with a combination of DHA, EPA, UMP, choline, iodide, zinc, and vitamin B12 may provide neuroprotection after perinatal HI.

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.