S Faulkner

Melatonin augments hypothermic neuroprotection in a perinatal asphyxia model

Despite treatment with therapeutic hypothermia, almost 50% of infants with neonatal encephalopathy still have adverse outcomes. Additional treatments are required to maximize neuroprotection. Melatonin is a naturally occurring hormone involved in physiological processes that also has neuroprotective actions against hypoxic-ischaemic brain injury in animal models. The objective of this study was to assess neuroprotective effects of combining melatonin with therapeutic hypothermia after transient hypoxia-ischaemia in a piglet model of perinatal asphyxia using clinically relevant magnetic resonance spectroscopy biomarkers supported by immunohistochemistry. After a quantified global hypoxic-ischaemic insult, 17 newborn piglets were randomized to the following: (i) therapeutic hypothermia (33.5°C from 2 to 26 h after resuscitation, n = 8) and (ii) therapeutic hypothermia plus intravenous melatonin (5 mg/kg/h over 6 h started at 10 min after resuscitation and repeated at 24 h, n = 9). Cortical white matter and deep grey matter voxel proton and whole brain (31)P magnetic resonance spectroscopy were acquired before and during hypoxia-ischaemia, at 24 and 48 h after resuscitation. There was no difference in baseline variables, insult severity or any physiological or biochemical measure, including mean arterial blood pressure and inotrope use during the 48 h after hypoxia-ischaemia. Plasma levels of melatonin were 10 000 times higher in the hypothermia plus melatonin than hypothermia alone group. Melatonin-augmented hypothermia significantly reduced the hypoxic-ischaemic-induced increase in the area under the curve for proton magnetic resonance spectroscopy lactate/N-acetyl aspartate and lactate/total creatine ratios in the deep grey matter. Melatonin-augmented hypothermia increased levels of whole brain (31)P magnetic resonance spectroscopy nucleotide triphosphate/exchangeable phosphate pool. Correlating with improved cerebral energy metabolism, TUNEL-positive nuclei were reduced in the hypothermia plus melatonin group compared with hypothermia alone in the thalamus, internal capsule, putamen and caudate, and there was reduced cleaved caspase 3 in the thalamus. Although total numbers of microglia were not decreased in grey or white matter, expression of the prototypical cytotoxic microglial activation marker CD86 was decreased in the cortex at 48 h after hypoxia-ischaemia. The safety and improved neuroprotection with a combination of melatonin with cooling support phase II clinical trials in infants with moderate and severe neonatal encephalopathy.

Xenon augmented hypothermia reduces early lactate/N-acetylaspartate and cell death in perinatal asphyxia

Additional treatments for therapeutic hypothermia are required to maximize neuroprotection for perinatal asphyxial encephalopathy. We assessed neuroprotective effects of combining inhaled xenon with therapeutic hypothermia after transient cerebral hypoxia–ischemia in a piglet model of perinatal asphyxia using magnetic resonance spectroscopy (MRS) biomarkers supported by immunohistochemistry.

Brain Cell Death Is Reduced With Cooling by 3.5°C to 5°C but Increased With Cooling by 8.5°C in a Piglet Asphyxia Model

Background and Purpose— In infants with moderate to severe neonatal encephalopathy, whole-body cooling at 33°C to 34°C for 72 hours is standard care with a number needed to treat to prevent a adverse outcome of 6 to 7. The precise brain temperature providing optimal neuroprotection is unknown. Methods— After a quantified global cerebral hypoxic-ischemic insult, 28 piglets aged <24 hours were randomized (each group, n=7) to (1) normothermia (38.5°C throughout) or whole-body cooling 2 to 26 hours after insult to (2) 35°C, (3) 33.5°C, or (4) 30°C. At 48 hours after hypoxia-ischemia, delayed cell death (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling and cleaved caspase 3) and microglial ramification (ionized calcium-binding adapter molecule 1) were evaluated. Results— At 48 hours after hypoxia-ischemia, substantial cerebral injury was found in the normothermia and 30°C hypothermia groups. However, with 35°C and 33.5°C cooling, a clear reduction in delayed cell death and microglial activation was observed in most brain regions (P<0.05), with no differences between 35°C and 33.5°C cooling groups. A protective pattern was observed, with U-shaped temperature dependence in delayed cell death in periventricular white matter, caudate nucleus, putamen, hippocampus, and thalamus. A microglial activation pattern was also seen, with inverted U-shaped temperature dependence in periventricular white matter, caudate nucleus, internal capsule, and hippocampus (all P<0.05). Conclusions— Cooling to 35°C (an absolute drop of 3.5°C as in therapeutic hypothermia protocols) or to 33.5°C provided protection in most brain regions after a cerebral hypoxic-ischemic insult in the newborn piglet. Although the relatively wide therapeutic range of a 3.5°C to 5°C drop in temperature reassured, overcooling (an 8.5°C drop) was clearly detrimental in some brain regions.

Brain mitochondrial oxidative metabolism during and after cerebral hypoxia-ischemia studied by simultaneous phosphorus magnetic-resonance and broadband near-infrared spectroscopy☆

Background Multimodal measurements combining broadband near-infrared spectroscopy (NIRS) and phosphorus magnetic resonance spectroscopy (31P MRS) assessed associations between changes in the oxidation state of cerebral mitochondrial cytochrome-c-oxidase (Δ[oxCCO]) and 31P metabolite peak-area ratios during and after transient cerebral hypoxia–ischemia (HI) in the newborn piglet. Methods Twenty-four piglets (aged < 24 h) underwent transient HI (inspired oxygen fraction 9% and bilateral carotid artery occlusion for ~ 20 min). Whole-brain 31P MRS and NIRS data were acquired every minute. Inorganic phosphate (Pi)/epp, phosphocreatine (PCr)/epp, and total nucleotide triphosphate (NTP)/epp were measured by 31P MRS and were plotted against Δ[oxCCO] during HI and recovery (epp = exchangeable phosphate pool = Pi + PCr + 2γ-NTP + β-NTP). Results During HI Δ[oxCCO], PCr/epp and NTP/epp declined and Pi/epp increased. Significant correlations were seen between 31P ratios and Δ[oxCCO]; during HI a threshold point was identified where the relationship between Δ[oxCCO] and both NTP/epp and Pi/epp changed significantly. Outcome at 48 h related to recovery of Δ[oxCCO] and 31P ratios 1 h post-HI (survived: 1-h NTP/epp 0.22 ± 0.02, Δ[oxCCO] − 0.29 ± 0.50 μM; died: 1-h NTP/epp 0.10 ± 0.04, Δ[oxCCO] − 2.41 ± 1.48 μM). Conclusions Both lowered Δ[oxCCO] and NTP/epp 1 h post-HI indicated mitochondrial impairment. Animals dying before 48 h had slower recovery of both Δ[oxCCO] and 31P ratios by 1 h after HI.

Methyl‐isobutyl amiloride reduces brain Lac/NAA, cell death and microglial activation in a perinatal asphyxia model

Na+/H+ exchanger (NHE) blockade attenuates the detrimental consequences of ischaemia and reperfusion in myocardium and brain in adult and neonatal animal studies. Our aim was to use magnetic resonance spectroscopy (MRS) biomarkers and immunohistochemistry to investigate the cerebral effects of the NHE inhibitor, methyl isobutyl amiloride (MIA) given after severe perinatal asphyxia in the piglet. Eighteen male piglets (aged < 24 h) underwent transient global cerebral hypoxia‐ischaemia and were randomized to (i) saline placebo; or (ii) 3 mg/kg intravenous MIA administered 10 min post‐insult and 8 hourly thereafter. Serial phosphorus‐31 (31P) and proton (1H) MRS data were acquired before, during and up to 48 h after hypoxia‐ischaemia and metabolite‐ratio time‐series Area under the Curve (AUC) calculated. At 48 h, histological and immunohistochemical assessments quantified regional tissue injury. MIA decreased thalamic lactate/N‐acetylaspartate and lactate/creatine AUCs (both p < 0.05) compared with placebo. Correlating with improved cerebral energy metabolism, transferase mediated biotinylated d‐UTP nick end‐labelling (TUNEL) positive cell density was reduced in the MIA group in cerebral cortex, thalamus and white matter (all p < 0.05) and caspase 3 immunoreactive cells were reduced in pyriform cortex and caudate nucleus (both p < 0.05). Microglial activation was reduced in pyriform and midtemporal cortex (both p < 0.05). Treatment with MIA starting 10 min after hypoxia‐ischaemia was neuroprotective in this perinatal asphyxia model.

A xenon recirculating ventilator for the newborn piglet: developing clinical applications of xenon for neonates.

Context The clinical applications of xenon for the neonate include both anaesthesia and neuroprotection. However, due to the limited natural availability of xenon, special equipment is required to administer and recapture the gas to develop xenon as a therapeutic agent. Objective In order to test the xenon recirculating ventilator for the application of neuroprotection in a preclinical trial, our primary objective was to test the efficiency, reliability and safety of administering 50% xenon for 24 h in hypoxic ischaemic piglets. Design A prospective observational study. Setting Institute for Womens Health, University College London, January 2008 to March 2008. Animals Four anaesthetised male piglets, less than 24 h old, underwent a global hypoxic ischaemic insult for approximately 25 min prior to switching to the xenon recirculating ventilator. Intervention Between 2 and 26 h after hypoxic ischaemia, anaesthetised piglets were administered a mixture of 50% xenon, air, oxygen and isoflurane. Main outcome measures The primary outcome measure was blood gas PaCO2 (kPa) and secondary outcome measure was xenon gas use (l h−1), over the 24-h duration of xenon administration. Results The xenon recirculating ventilator provided effective ventilation, automated control of xenon/air gas mixtures, and stable blood gas PaCO2 (4.5 to 6.3 kPa) for 24 h of ventilation with the xenon recirculating ventilator. Total xenon use was minimal at approximately 0.6 l h−1 at a cost of approximately &OV0556;8 h−1. Additional features included an isoflurane scavenger and bellows height alarm. Conclusion Stable gas delivery to a piglet with minimal xenon loss and analogue circuitry made the xenon recirculating ventilator easy to use and it could be modified for other large animals and noble gas mixtures. The technologies, safety and efficiency of xenon delivery in this preclinical system have been taken forward in the development of neonatal ventilators for clinical use in phase II clinical trials for xenon-augmented hypothermia and for xenon anaesthesia.

3-Dimensional modelling of chick embryo eye development and growth using high resolution magnetic resonance imaging

Magnetic resonance imaging (MRI) is a powerful tool for generating 3-dimensional structural and functional image data. MRI has already proven valuable in creating atlases of mouse and quail development. Here, we have exploited high resolution MRI to determine the parameters necessary to acquire images of the chick embryo eye. Using a 9.4 Tesla (400 MHz) high field ultra-shielded and refrigerated magnet (Bruker), MRI was carried out on paraformaldehyde-fixed chick embryos or heads at E4, E6, E8, and E10. Image data were processed using established and custom packages (MRICro, ImageJ, ParaVision, Bruker and mri3dX). Voxel dimensions ranged from 62.5 microm to 117.2 microm. We subsequently used the images obtained from the MRI data in order to make precise measurements of chick embryo eye surface area, volume and axial length from E4 to E10. MRI was validated for accurate sizing of ocular tissue features by direct comparison with previously published literature. Furthermore, we demonstrate the utility of high resolution MRI for making accurate measurements of morphological changes due to experimental manipulation of chick eye development, thereby facilitating a better understanding of the effects on chick embryo eye development and growth of such manipulations. Chondroitin sulphate or heparin were microinjected into the vitreous cavity of the right eyes of each of 3 embryos at E5. At E10, embryos were fixed and various eye parameters (volume, surface area, axial length and equatorial diameter) were determined using MRI and normalised with respect to the un-injected left eyes. Statistically significant alterations in eye volume (p < 0.05; increases with chondroitin sulphate and decreases with heparin) and changes in vitreous homogeneity were observed in embryos following microinjection of glycosaminoglycans. Furthermore, in the heparin-injected eyes, significant disturbances at the vitreo-retinal boundary were observed as well as retinal folding and detachment confirming histological observations. These data reveal the utility and superiority of MRI for producing images enabling quantification of experimentally induced changes in eye volume and structure. The results indicate that MRI is an important tool that could become a routine approach for rapid and sensitive phenotypic analysis of normal chick ocular development and morphology as well as potentially the effects of surgical or genetic manipulations of chick embryo eyes in live embryos in ovo.

Pharmacokinetics of dexmedetomidine combined with therapeutic hypothermia in a piglet asphyxia model

The highly selective α2‐adrenoreceptor agonist, dexmedetomidine, exerts neuroprotective, analgesic, anti‐inflammatory and sympatholytic properties that may be beneficial for perinatal asphyxia. The optimal safe dose for pre‐clinical newborn neuroprotection studies is unknown.

Optimal Wavelength Combinations for Resolving in-vivo Changes of Haemoglobin and Cytochrome-c-oxidase Concentrations with NIRS

Novel method used to identify the optimal three and four wavelength combinations (out of 120, 780-900nm) to accurately resolve the concentration changes in haemoglobin and cytochrome-c-oxidase during hypoxic-ischaemia in a neonatal preclinical model.

Simulating NIRS and MRS Measurements During Cerebral Hypoxia-Ischaemia in Piglets Using a Computational Model

We present a group analysis of the changes in cerebral haemodynamics, and the oxidation state of cytochrome-c-oxidase measured using broadband near-infrared spectroscopy (NIRS) and intracellular pH measured by phosphorous (31P) magnetic resonance spectroscopy (MRS) during and after cerebral hypoxia-ischaemia (HI) in 15 piglets. We use a previously published computational model of cerebral metabolism in the piglet [1] to integrate these measurements and simulate HI. We successfully simulate changes in cellular metabolism including shifts in intracellular pH observed in the piglet brain during HI. In this process, we optimise physiological parameters in the model identified through sensitivity analysis (such as the rate of glucose metabolism and intracellular lactate concentration), to fit simulated and measured data. The model fits the data reasonably and suggests a 20 % drop in glucose consumption, a ~65 % increase in lactate concentration and ~35 % drop in the cerebral metabolic rate of oxygen (CMRO2) during HI.