Dexmedetomidine Combined with Therapeutic Hypothermia Is Associated with Cardiovascular Instability and Neurotoxicity in a Piglet Model of Perinatal Asphyxia.

The selective α2-adrenoreceptor agonist dexmedetomidine has shown neuroprotective, analgesic, anti-inflammatory, and sympatholytic properties that may be beneficial in neonatal encephalopathy (NE). As therapeutic hypothermia is only partially effective, adjunct therapies are needed to optimize outcomes. The aim was to assess whether hypothermia + dexmedetomidine treatment augments neuroprotection compared to routine treatment (hypothermia + fentanyl sedation) in a piglet model of NE using magnetic resonance spectroscopy (MRS) biomarkers, which predict outcomes in babies with NE, and immunohistochemistry. After hypoxia-ischaemia (HI), 20 large White male piglets were randomized to: (i) hypothermia + fentanyl with cooling to 33.5°C from 2 to 26 h, or (ii) hypothermia + dexmedetomidine (a loading dose of 2 µg/kg at 10 min followed by 0.028 µg/kg/h for 48 h). Whole-brain phosphorus-31 and regional proton MRS biomarkers were assessed at baseline, 24, and 48 h after HI. At 48 h, cell death was evaluated over 7 brain regions by means of transferase-mediated d-UTP nick end labeling (TUNEL). Dexmedetomidine plasma levels were mainly within the target sedative range of 1 µg/L. In the hypothermia + dexmedetomidine group, there were 6 cardiac arrests (3 fatal) versus 2 (non-fatal) in the hypothermia + fentanyl group. The hypothermia + dexmedetomidine group required more saline (p = 0.005) to maintain blood pressure. Thalamic and white-matter lactate/N-acetylaspartate did not differ between groups (p = 0.66 and p = 0.21, respectively); the whole-brain nucleotide triphosphate/exchangeable phosphate pool was similar (p = 0.73) over 48 h. Cell death (TUNEL-positive cells/mm2) was higher in the hypothermia + dexmedetomidine group than in the hypothermia + fentanyl group (mean 5.1 vs. 2.3, difference 2.8 [95% CI 0.6-4.9], p = 0.036). Hypothermia + dexmedetomidine treatment was associated with adverse cardiovascular events, even within the recommended clinical sedative plasma level; these may have been exacerbated by an interaction with either isoflurane or low body temperature. Hypothermia + dexmedetomidine treatment was neurotoxic following HI in our piglet NE model, suggesting that caution is vital if dexmedetomidine is combined with cooling following NE.

Surgery increases cell death and induces changes in gene expression compared with anesthesia alone in the developing piglet brain.

In a range of animal species, exposure of the brain to general anaesthesia without surgery during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex but include an increase in brain cell death. In humans, attempts to link adverse cognitive development to infantile anaesthesia exposure have yielded ambiguous results. One caveat that may influence the interpretation of human studies is that infants are not exposed to general anaesthesia without surgery, raising the possibility that surgery itself, may contribute to adverse cognitive development. Using piglets, we investigated whether a minor surgical procedure increases cell death and disrupts neuro-developmental and cognitively salient gene transcription in the neonatal brain. We randomly assigned neonatal male piglets to a group who received 6h of 2% isoflurane anaesthesia or a group who received an identical anaesthesia plus 15 mins of surgery designed to replicate an inguinal hernia repair. Compared to anesthesia alone, surgery-induced significant increases in cell death in eight areas of the brain. Using RNAseq data derived from all 12 piglets per group we also identified significant changes in the expression of 181 gene transcripts induced by surgery in the cingulate cortex, pathway analysis of these changes suggests that surgery influences the thrombin, aldosterone, axonal guidance, B cell, ERK-5, eNOS and GABAA signalling pathways. This suggests a number of novel mechanisms by which surgery may influence neural and cognitive development independently or synergistically with the effects of anaesthesia.

Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain.

Exposure of the brain to general anesthesia during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex, incompletely understood and may be sexually dimorphic, but include developmentally inappropriate apoptosis, inflammation and a disruption to cognitively salient gene expression. We investigated the effects of a 6h isoflurane exposure on cell death, microglial activation and gene expression in the male neonatal piglet brain. Piglets (n = 6) were randomised to: (i) naive controls or (ii) 6h isoflurane. Cell death (TUNEL and caspase-3) and microglial activation were recorded in 7 brain regions. Changes in gene expression (microarray and qPCR) were assessed in the cingulate cortex. Electroencephalography (EEG) was recorded throughout. Isoflurane anesthesia induced significant increases in cell death in the cingulate and insular cortices, caudate nucleus, thalamus, putamen, internal capsule, periventricular white matter and hippocampus. Dying cells included both neurons and oligodendrocytes. Significantly, microglial activation was observed in the insula, pyriform, hippocampus, internal capsule, caudate and thalamus. Isoflurane induced significant disruption to the expression of 79 gene transcripts, of these 26 are important for the control of transcription and 23 are important for the mediation of neural plasticity, memory formation and recall. Our observations confirm that isoflurane increases apoptosis and inflammatory responses in the neonatal piglet brain but also suggests novel additional mechanisms by which isoflurane may induce adverse neural and cognitive development by disrupting the expression of genes mediating activity dependent development of neural circuits, the predictive adaptive responses of the brain, memory formation and recall.

Immediate remote ischemic postconditioning after hypoxia ischemia in piglets protects cerebral white matter but not grey matter.

Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention whereby brief episodes of ischemia/reperfusion of one organ (limb) mitigate damage in another organ (brain) that has experienced severe hypoxia-ischemia. Our aim was to assess whether RIPostC is protective following cerebral hypoxia-ischemia in a piglet model of neonatal encephalopathy (NE) using magnetic resonance spectroscopy (MRS) biomarkers and immunohistochemistry. After hypoxia-ischemia (HI), 16 Large White female newborn piglets were randomized to: (i) no intervention (n = 8); (ii) RIPostC - with four, 10-min cycles of bilateral lower limb ischemia/reperfusion immediately after HI (n = 8). RIPostC reduced the hypoxic-ischemic-induced increase in white matter proton MRS lactate/N acetyl aspartate (p = 0.005) and increased whole brain phosphorus-31 MRS ATP (p = 0.039) over the 48 h after HI. Cell death was reduced with RIPostC in the periventricular white matter (p = 0.03), internal capsule (p = 0.002) and corpus callosum (p = 0.021); there was reduced microglial activation in corpus callosum (p = 0.001) and more surviving oligodendrocytes in corpus callosum (p = 0.029) and periventricular white matter (p = 0.001). Changes in gene expression were detected in the white matter at 48 h, including KATP channel and endothelin A receptor. Immediate RIPostC is a potentially safe and promising brain protective therapy for babies with NE with protection in white but not grey matter.

Inhaled 45-50% argon augments hypothermic brain protection in a piglet model of perinatal asphyxia.

Cooling to 33.5°C in babies with neonatal encephalopathy significantly reduces death and disability, however additional therapies are needed to maximize brain protection. Following hypoxia-ischemia we assessed whether inhaled 45-50% Argon from 2-26h augmented hypothermia neuroprotection in a neonatal piglet model, using MRS and aEEG, which predict outcome in babies with neonatal encephalopathy, and immunohistochemistry. Following cerebral hypoxia-ischemia, 20 Newborn male Large White piglets<40h were randomized to: (i) Cooling (33°C) from 2-26h (n=10); or (ii) Cooling and inhaled 45-50% Argon (Cooling+Argon) from 2-26h (n=8). Whole-brain phosphorus-31 and regional proton MRS were acquired at baseline, 24 and 48h after hypoxia-ischemia. EEG was monitored. At 48h after hypoxia-ischemia, cell death (TUNEL) was evaluated over 7 brain regions. There were no differences in body weight, duration of hypoxia-ischemia or insult severity; throughout the study there were no differences in heart rate, arterial blood pressure, blood biochemistry and inotrope support. Two piglets in the Cooling+Argon group were excluded. Comparing Cooling+Argon with Cooling there was preservation of whole-brain MRS ATP and PCr/Pi at 48h after hypoxia-ischemia (p<0.001 for both) and lower (1)H MRS lactate/N acetyl aspartate in white (p=0.03 and 0.04) but not gray matter at 24 and 48h. EEG background recovery was faster (p<0.01) with Cooling+Argon. An overall difference between average cell-death of Cooling versus Cooling+Argon was observed (p<0.01); estimated cells per mm(2) were 23.9 points lower (95% C.I. 7.3-40.5) for the Cooling+Argon versus Cooling. Inhaled 45-50% Argon from 2-26h augmented hypothermic protection at 48h after hypoxia-ischemia shown by improved brain energy metabolism on MRS, faster EEG recovery and reduced cell death on TUNEL. Argon may provide a cheap and practical therapy to augment cooling for neonatal encephalopathy.

New horizons for newborn brain protection: enhancing endogenous neuroprotection.

Intrapartum-related events are the third leading cause of childhood mortality worldwide and result in one million neurodisabled survivors each year. Infants exposed to a perinatal insult typically present with neonatal encephalopathy (NE). The contribution of pure hypoxia-ischaemia (HI) to NE has been debated; over the last decade, the sensitising effect of inflammation in the aetiology of NE and neurodisability is recognised. Therapeutic hypothermia is standard care for NE in high-income countries; however, its benefit in encephalopathic babies with sepsis or in those born following chorioamnionitis is unclear. It is now recognised that the phases of brain injury extend into a tertiary phase, which lasts for weeks to years after the initial insult and opens up new possibilities for therapy.There has been a recent focus on understanding endogenous neuroprotection and how to boost it or to supplement its effectors therapeutically once damage to the brain has occurred as in NE. In this review, we focus on strategies that can augment the body's own endogenous neuroprotection. We discuss in particular remote ischaemic postconditioning whereby endogenous brain tolerance can be activated through hypoxia/reperfusion stimuli started immediately after the index hypoxic-ischaemic insult. Therapeutic hypothermia, melatonin, erythropoietin and cannabinoids are examples of ways we can supplement the endogenous response to HI to obtain its full neuroprotective potential. Achieving the correct balance of interventions at the correct time in relation to the nature and stage of injury will be a significant challenge in the next decade.

Modelling blood flow and metabolism in the piglet brain during hypoxia-ischaemia: simulating pH changes.

We describe the extension of a computational model of blood flow and metabolism in the piglet brain to investigate changes in neonatal intracellular brain pH during hypoxia-ischemia (HI). The model is able to simulate near-infrared spectroscopy (NIRS) and magnetic resonance spectroscopy (MRS) measurements obtained from HI experiments conducted in piglets. We adopt a method of using (31)P-MRS data to estimate of intracellular pH and compare measured pH and oxygenation with their modelled counterparts. We show that both NIRS and MRS measurements are predicted well in the new version of the model.

Modelling blood flow and metabolism in the piglet brain during hypoxia-ischaemia: simulating brain energetics.

We have developed a computational model to simulate hypoxia-ischaemia (HI) in the neonatal piglet brain. It has been extended from a previous model by adding the simulation of carotid artery occlusion and including pH changes in the cytoplasm. Here, simulations from the model are compared with near-infrared spectroscopy (NIRS) and phosphorus magnetic resonance spectroscopy (MRS) measurements from two piglets during HI and short-term recovery. One of these piglets showed incomplete recovery after HI, and this is modelled by considering some of the cells to be dead. This is consistent with the results from MRS and the redox state of cytochrome-c-oxidase as measured by NIRS. However, the simulations do not match the NIRS haemoglobin measurements. The model therefore predicts that further physiological changes must also be taking place if the hypothesis of dead cells is correct.