Small animal PET with spontaneous inhalation of 15O-labelled oxygen gases: Longitudinal assessment of cerebral oxygen metabolism in a rat model of neonatal hypoxic-ischaemic encephalopathy.

Perinatal hypoxic-ischaemic encephalopathy (HIE) is the leading cause of irreversible brain damage resulting in serious neurological dysfunction among neonates. We evaluated the feasibility of positron emission tomography (PET) methodology with 15O-labelled gases without intravenous or tracheal cannulation for assessing temporal changes in cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMRO2) in a neonatal HIE rat model. Sequential PET scans with spontaneous inhalation of 15O-gases mixed with isoflurane were performed over 14 days after the hypoxic-ischaemic insult in HIE pups and age-matched controls. CBF and CMRO2 in the injured hemispheres of HIE pups remarkably decreased 2 days after the insult, gradually recovering over 14 days in line with their increase found in healthy controls according to their natural maturation process. The magnitude of hemispheric tissue loss histologically measured after the last PET scan was significantly correlated with the decreases in CBF and CMRO2.This fully non-invasive imaging strategy may be useful for monitoring damage progression in neonatal HIE and for evaluating potential therapeutic outcomes.

Preclinical assessment of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells for neonatal hypoxic-ischaemic brain injury.

Hypoxic-ischaemic encephalopathy (HIE) is a type of brain injury affecting approximately 1 million newborn babies per year worldwide, the only treatment for which is therapeutic hypothermia. Thrombin-preconditioned mesenchymal stem cells (MSCs) exert neuroprotective effects by enriching cargo contents and boosting exosome biogenesis, thus showing promise as a new therapeutic strategy for HIE. This study was conducted to evaluate the tissue distribution and potential toxicity of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells (th-hWJMSCs) in animal models before the initiation of clinical trials. We investigated the biodistribution, tumorigenicity and general toxicity of th-hWJMSCs. MSCs were administered the maximum feasible dose (1 × 105 cells/10 µL/head) once, or at lower doses into the cerebral ventricle. To support the clinical use of th-hWJMSCs for treating brain injury, preclinical safety studies were conducted in newborn Sprague-Dawley rats and BALB/c nude mice. In addition, growth parameters were evaluated to assess the impact of th-hWJMSCs on the growth of newborn babies. Our results suggest that th-hWJMSCs are non-toxic and non-tumorigenic in rodent models, survive for up to 7 days in the brain and hold potential for HIE therapy.

Magnetic resonance spectroscopy assessment of brain injury after moderate hypothermia in neonatal encephalopathy: a prospective multicentre cohort study.

BACKGROUND: In neonatal encephalopathy, the clinical manifestations of injury can only be reliably assessed several years after an intervention, complicating early prognostication and rendering trials of promising neuroprotectants slow and expensive. We aimed to determine the accuracy of thalamic proton magnetic resonance (MR) spectroscopy (MRS) biomarkers as early predictors of the neurodevelopmental abnormalities observed years after neonatal encephalopathy. METHODS: We did a prospective multicentre cohort study across eight neonatal intensive care units in the UK and USA, recruiting term and near-term neonates who received therapeutic hypothermia for neonatal encephalopathy. We excluded infants with life-threatening congenital malformations, syndromic disorders, neurometabolic diseases, or any alternative diagnoses for encephalopathy that were apparent within 6 h of birth. We obtained T1-weighted, T2-weighted, and diffusion-weighted MRI and thalamic proton MRS 4-14 days after birth. Clinical neurodevelopmental tests were done 18-24 months later. The primary outcome was the association between MR biomarkers and an adverse neurodevelopmental outcome, defined as death or moderate or severe disability, measured using a multivariable prognostic model. We used receiver operating characteristic (ROC) curves to examine the prognostic accuracy of the individual biomarkers. This trial is registered with ClinicalTrials.gov, number NCT01309711. FINDINGS: Between Jan 29, 2013, and June 25, 2016, we recruited 223 infants who all underwent MRI and MRS at a median age of 7 days (IQR 5-10), with 190 (85%) followed up for neurological examination at a median age of 23 months (20-25). Of those followed up, 31 (16%) had moderate or severe disability, including one death. Multiple logistic regression analysis could not be done because thalamic N-acetylaspartate (NAA) concentration alone accurately predicted an adverse neurodevelopmental outcome (area under the curve [AUC] of 0·99 [95% CI 0·94-1·00]; sensitivity 100% [74-100]; specificity 97% [90-100]; n=82); the models would not converge when any additional variable was examined. The AUC (95% CI) of clinical examination at 6 h (n=190) and at discharge (n=167) were 0·72 (0·65-0·78) and 0·60 (0·53-0·68), respectively, and the AUC of abnormal amplitude integrated EEG at 6 h (n=169) was 0·73 (0·65-0·79). On conventional MRI (n=190), cortical injury had an AUC of 0·67 (0·60-0·73), basal ganglia or thalamic injury had an AUC of 0·81 (0·75-0·87), and abnormal signal in the posterior limb of internal capsule (PLIC) had an AUC of 0·82 (0·76-0·87). Fractional anisotropy of PLIC (n=65) had an AUC of 0·82 (0·76-0·87). MRS metabolite peak-area ratios (n=160) of NAA-creatine (<1·29) had an AUC of 0·79 (0·72-0·85), of NAA-choline had an AUC of 0·74 (0·66-0·80), and of lactate-NAA (>0·22) had an AUC of 0·94 (0·89-0·97). INTERPRETATION: Thalamic proton MRS measures acquired soon after birth in neonatal encephalopathy had the highest accuracy to predict neurdevelopment 2 years later. These methods could be applied to increase the power of neuroprotection trials while reducing their duration. FUNDING: National Institute for Health Research UK.

Population Pharmacokinetics of Gentamicin in Neonates with Hypoxemic-Ischemic Encephalopathy Receiving Controlled Hypothermia.

OBJECTIVE: Identify population pharmacokinetics and pharmacodynamic target attainment of gentamicin in neonates with hypoxic-ischemic encephalopathy (HIE) undergoing controlled hypothermia (CH). DESIGN: Prospective open-label pharmacokinetic study. Gentamicin concentrations were modeled and dosing regimens simulated for a 5000-patient neonatal population with HIE receiving CH using PMetrics, a nonparametric, pharmacometric modeling, and simulation package for R. SETTING: A 189-bed children's tertiary care teaching hospital. RESULTS: Twelve patients, 5 (42%) females and 7 (58%) males, met inclusion criteria with a median gestation age of 39.9 weeks (interquartile range [IQR] 38.5-40.2 wks) and a median birthweight (BW) of 3.3 kg (IQR 3.1-3.7 kg). Gentamicin concentrations were best described by a two-compartment model with first-order elimination with BW as a covariate on volume of distribution (Vd). The mean total body population clearance (CL) was 2.2 ± 0.7 ml/minute/kg, and the volume of the central compartment was 0.44 ± 0.06 L/kg. The R2 , bias, and precision for the observed versus population predicted model were 0.917, 1.15, and 10.9 µg/ml; the R2 , bias, and precision for the observed versus individual predicted model were 0.982, -0.132, and 0.932 µg/ml, respectively. The calculated mean population estimate for the total Vd was 0.96 ± 0.4 L/kg. The dosing regimen that most consistently produced a maximum concentration (Cmax ) in the range of 10-12 mg/L with a minimum concentration (Cmin ) level less than 2 mg/L was 5 mg/kg/dose given every 36 hours. CONCLUSION: These data suggest the population pharmacokinetics of gentamicin in neonates with HIE receiving CH have an increase in gentamicin CL and are different from previous reports in neonates with HIE not receiving CH and/or neonates without HIE. This analysis suggests a dosing regimen of 5 mg/kg/dose every 36 hours results in a gentamicin Cmax within the range of 10-12 mg/L with a Cmin lower than 2 mg/L, which is appropriate for treating susceptible gram-negative organisms with minimum inhibitory concentrations of 1 mg/L or lower.

Assessment of long-term safety and efficacy of intranasal mesenchymal stem cell treatment for neonatal brain injury in the mouse.

BACKGROUND: For clinical translation, we assessed whether intranasal mesenchymal stem cell (MSC) treatment after hypoxia-ischemia (HI) induces neoplasia in the brain or periphery at 14 mo. Furthermore, the long-term effects of MSCs on behavior and lesion size were determined. METHOD: HI was induced in 9-d-old mice. Pups received an intranasal administration of 0.5 × 10(6) MSCs or vehicle at 10 d post-HI. Full macroscopical and microscopical pathological analysis of 39 organs per mouse was performed. Sensorimotor behavior was assessed in the cylinder-rearing test at 10 d, 28 d, 6 mo, and 9 mo. Cognition was measured with the novel object recognition test at 3 and 14 mo post-HI. Lesion size was determined by analyzing mouse-anti-microtubule-associated protein 2 (MAP2) and mouse-anti-myelin basic protein (MBP) staining at 5 wk and 14 mo. RESULTS: At 14 mo post-HI, we did not observe any neoplasia in the nasal turbinates, brain, or other organs of HI mice treated with MSCs. Furthermore, our results show that MSC-induced improvement of sensorimotor and cognitive function is long lasting. In contrast, HI-vehicle mice showed severe behavioral impairment. Recovery of MAP2- and MBP-positive area lasted up to 14 mo following MSC treatment. CONCLUSION: Our results provide strong evidence of the long-term safety and positive effects of MSC treatment following neonatal HI in mice.

[Assessment of therapeutic passive hypothermia in newborns with hypoxic-ischemic encephalopathy that need interhospital transport]. / Valoración de la hipotermia terapéutica pasiva del recién nacido con encefalopatía hipóxico-isquémica que precisa traslado interhospitalario.

INTRODUCTION: Induced hypothermia for the first hours of life in a newborn is an effective treatment to reduce mortality and serious effects in neonates that had suffered a hypoxia episode. This method needs an universal attendance independently of the place of birth being usually necessary a transfer to the reference hospital. AIM: To analyze the efficacy of the newborn with hypoxic-ischemic encephalopathy transfer in passive hypothermia. PATIENTS AND METHODS: Descriptive study of series of cases with retrospective character of newborn from Cadiz's province that need induced hypothermia. 46 newborn were included in the study: 33 of them (71.74%) needed being transfer by the Critical Patients Transport service (CPT group), the rest (28.26%) were born into the reference hospital. RESULTS: Both groups are similar in age gestational at birth, sex, weight and hypoxic-ischemic encephalopathy degree. It analyzed variables related to hypothermia therapy and in addition in CPT group transfer specific variables. At discharge, it does not exist significant differences between groups in the efficiency-consequence of neuroprotection therapy with hypothermia (p = 0.159). It does not find complications derived from the interhospital move. CONCLUSIONS: Neonatal inter-hospital transfer in passive therapeutic hypothermia is effective, safe and necessary for the therapy compliance. It is required reach an agreement between the attendance and the reference service, setting up guides for the support and suitable range of temperature.

TITLE: Valoracion de la hipotermia terapeutica pasiva del recien nacido con encefalopatia hipoxico-isquemica que precisa traslado interhospitalario.Introduccion. La hipotermia inducida durante las primeras horas de vida del recien nacido es un tratamiento eficaz para reducir la mortalidad y secuelas graves en neonatos que han sufrido un episodio de hipoxia perinatal. Este procedimiento requiere una asistencia universalizada independiente del centro donde haya nacido, siendo necesario su traslado al hospital de referencia. Objetivo. Evaluar la eficacia del traslado interhospitalario del neonato con encefalopatia hipoxico-isquemica en hipotermia pasiva. Pacientes y metodos. Estudio descriptivo de series de casos con caracter retrospectivo. Se estudiaron neonatos de la provincia de Cadiz que precisaron hipotermia inducida. Se incluyo a un total de 46 neonatos en el analisis: 33 de ellos (71,74%) precisaron traslado por el Servicio de Traslados de Pacientes Criticos (grupo TPC); el resto (28,26%) nacio en el centro de referencia. Resultados. Ambos grupos son comparables en edad gestacional al nacimiento, sexo, peso y grado de encefalopatia hipoxico-isquemica. Se analizan variables relacionadas con la aplicacion de la hipotermia, y en el grupo TPC se analizan variables relacionadas con el traslado. No se aprecian diferencias significativas entre los grupos en la efectividad-consecuencia de la terapia neuroprotectora con hipotermia al alta hospitalaria (p = 0,159). No se encuentran complicaciones derivadas del traslado interhospitalario. Conclusiones. El traslado interhospitalario del neonato en hipotermia terapeutica realizado de forma pasiva es efectivo, seguro y necesario para el cumplimiento de la terapia. Es preciso consensuar la asistencia con el servicio de referencia, estableciendo guias en cuanto al soporte y rango de temperatura adecuada, consolidando asi una asistencia integral.

Multi-modal assessment of long-term erythropoietin treatment after neonatal hypoxic-ischemic injury in rat brain.

Erythropoietin (EPO) has been recognized as a neuroprotective agent. In animal models of neonatal brain injury, exogenous EPO has been shown to reduce lesion size, improve structure and function. Experimental studies have focused on short course treatment after injury. Timing, dose and length of treatment in preterm brain damage remain to be defined. We have evaluated the effects of high dose and long-term EPO treatment in hypoxic-ischemic (HI) injury in 3 days old (P3) rat pups using histopathology, magnetic resonance imaging (MRI) and spectroscopy (MRS) as well as functional assessment with somatosensory-evoked potentials (SEP). After HI, rat pups were assessed by MRI for initial damage and were randomized to receive EPO or vehicle. At the end of treatment period (P25) the size of resulting cortical damage and white matter (WM) microstructure integrity were assessed by MRI and cortical metabolism by MRS. Whisker elicited SEP were recorded to evaluate somatosensory function. Brains were collected for neuropathological assessment. The EPO treated animals did not show significant decrease of the HI induced cortical loss at P25. WM microstructure measured by diffusion tensor imaging was improved and SEP response in the injured cortex was recovered in the EPO treated animals compared to vehicle treated animals. In addition, the metabolic profile was less altered in the EPO group. Long-term treatment with high dose EPO after HI injury in the very immature rat brain induced recovery of WM microstructure and connectivity as well as somatosensory cortical function despite no effects on volume of cortical damage. This indicates that long-term high-dose EPO induces recovery of structural and functional connectivity despite persisting gross anatomical cortical alteration resulting from HI.

Assessment of brain oxygenation in term and preterm neonates using near infrared spectroscopy.

PURPOSE: The aim of this study was to determine brain oxygenation in full-term and preterm neonates using near infrared spectroscopy. MATERIAL AND METHODS: A total of 88 full-term and preterm newborn infants without hypoxic-ischaemic disorders admitted to the NICU were examined using NIRS on the first day of life and on day 28 of life. Additional measurements were taken at the end of the first week of life in the premature neonates group. Measurements of oxyhaemoglobin (HbO2), deoxyhaemoglobin (Hb), total haemoglobin (HbT) concentration and tissue oxygen saturation (Ox) were performed in 5 brain regions. Right and left frontal areas, the occipital area and right and left temporal areas were measured. RESULTS: In full-term healthy neonates a marked decrease in HbO, Hb and HbT values was observed on day 28 of life in all brain regions except the occipital area. In the neonatal period the greatest changes in brain oxygenation occurred in the right and left frontal regions of the brain. In preterm neonates constant values of HbO2 and Ox were observed in the first 28 days of life. In preterm newborn infants, as well as in full term newborn infants, similar Ox and HbO2 values were obtained on day 28 of life. CONCLUSIONS: NIRS is a safe method and can be used to evaluate brain oxygenation in newborn infants. The results of these measurements are in accordance with changes in brain oxygenation in the first month of life, which are predicated on the basis of the neonate's physiology.

An isolation method for assessment of brain mitochondria function in neonatal mice with hypoxic-ischemic brain injury.

This work was undertaken to develop a method for the isolation of mitochondria from a single cerebral hemisphere in neonatal mice. Mitochondria from the normal mouse brain hemisphere isolated by the proposed method exhibited a good respiratory control ratio of 6.39 +/- 0.53 during glutamate-malate-induced phosphorylating respiration. Electron microscopy showed intact mitochondria. The applicability of this method was tested on mitochondria isolated from naïve mice and their littermates subjected to hypoxic-ischemic insult. Hypoxic-ischemic insult prior to reperfusion resulted in a significant (p < 0.01) inhibition of phosphorylating respiration compared to naïve littermates. This was associated with a profound depletion of the ATP content in the ischemic hemisphere. The expression for Mn superoxide dismutase and cytochrome C (markers for the integrity of the mitochondrial matrix and outer membrane) was determined by Western blot to control for mitochondrial integrity and quantity in the compared samples. Thus, we have developed a method for the isolation of the cerebral mitochondria from a single hemisphere adapted to neonatal mice. This method may serve as a valuable tool to study mitochondrial function in a mouse model of immature brain injury. In addition, the suggested method enables us to examine the mitochondrial functional phenotype in immature mice with a targeted genetic alteration.

[Early assessment of severe hypoxic-ischemic encephalopathy in neonates by diffusion-weighted magnetic resonance imaging techniques and its significance].

OBJECTIVE: Hypoxic-ischemic encephalopathy (HIE) is an important cause of morbidity and mortality in the neonates. Early and accurate diagnosis is helpful not only for assessing prognosis but also for making treatment decisions. The aim of this study was to explore the value of early assessment of HIE by applying the diffusion-weighted imaging (DWI) in acute (within 72 hours), subacute or chronic stages of HIE in comparison to conventional magnetic resonance imaging (MRI) in clinical practice. METHODS: Images and clinical charts of fourteen term neonates with clinically diagnosed severe hypoxic-ischemic encephalopathy treated in the NICU from January 2006 to February 2007 were retrospectively reviewed. Inclusion criteria were: term infant (37 approximately 42 weeks) and high clinical suspicion of severe HIE (low Apgar scores, need for resuscitation, metabolic acidosis, acute encephalopathy (eg, hypotonia, coma, seizures). All examinations were performed on a 3.0-T MRI system (Philips Intera Acheva Magnetom Vision) with echo-planar imaging capability with the use of a standard protocol. The imaging protocol for all the patients contained diffuse weighted images (EPI-SE, TR = 2144 ms, TE = 56 ms), T1-weighted images (TR = 389 ms; TE = 15 ms; slice thickness = 4 mm) as well as T2-weighted images (TR = 3035 ms; TE = 100 ms; slice thickness = 4 mm). The studies were first performed within 72 hours of life in these 14 consecutive patients, including both standard T1, T2-weighted image and DWI; follow-up MR studies were performed for 4 patients at the ages of 7 days, for 4 at 14 days, for another 3 at ages of both 21 days and 8 months. RESULTS: First inspection (on an average of 48 hours after birth): routine T1, T2-weighted images showed normal images in all patients, while diffusion images showed symmetric high intensity signal in the lateral thalami and posterior limbs of internal capsules (PLIC). Following up: on day 7, routine MRI showed both symmetric T1 prolongation and T2 slightly shortening in lateral thalami, DWI showed abnormal high signal intensity in bilateral basal ganglion (mainly in the back site of lentiform nuclei, putamen) and the cortex around central sulcus, but the previous hyperintensity in lateral thalami and PLIC disappeared. On day 14, routine MRI showed symmetric T1 prolongation, T2 shortening in bilateral thalami, lentiform nuclei and cortex around central sulus. On day 21, routine MRI showed T1 prolongation, T2 shortening in bilateral thalami and basal ganglion while previously obvious PLIC disappeared, whereas DWI showed normal images. Eight months later, deeper cerebral sulus, dilation of ventricles and widening of extracerebral space were shown. CONCLUSION: Diffusion-weighted imaging has proved more sensitive than conventional MR imaging sequences in detecting acute cerebral infarction in adult subjects. DWI is proposed as a method for early detection of hypoxic-ischemic brain injury. In this study, DWI showed the same focus (lateral thalami and PLIC) and similar extent of the injury in these severe HIE patients in the early stage after birth (in 72 hours). The sites which showed hyperintensive signals in DWI were consistent with the foci in subsequent follow-up by routine MRI. Thus, DWI is supposed to be a technique for early assessment of the extent of hypoxic-ischemic brain injury and the prognosis in clinic. Though DWI is superior to the other imaging modalities in detecting ischemia, diffusion restriction is not necessarily indicative of permanent damage. The abnormal image on DWI may not last long. However, in chronic stage, the follow-up conventional MRI may compensate the inadequacy of DWI.

Is anoxic depolarisation associated with an ADC threshold? A Markov chain Monte Carlo analysis.

A Bayesian nonlinear hierarchical random coefficients model was used in a reanalysis of a previously published longitudinal study of the extracellular direct current (DC)-potential and apparent diffusion coefficient (ADC) responses to focal ischaemia. The main purpose was to examine the data for evidence of an ADC threshold for anoxic depolarisation. A Markov chain Monte Carlo simulation approach was adopted. The Metropolis algorithm was used to generate three parallel Markov chains and thus obtain a sampled posterior probability distribution for each of the DC-potential and ADC model parameters, together with a number of derived parameters. The latter were used in a subsequent threshold analysis. The analysis provided no evidence indicating a consistent and reproducible ADC threshold for anoxic depolarisation.

Assessment of the hypoxic-ischemic encephalopathy in neonates using non-invasive near-infrared spectroscopy.

This paper introduces a method of monitoring cerebral oxygenation for healthy neonates and neonates with hypoxic-ischemic encephalopathy (HIE) using near-infrared spectroscopy. The object of this study was to investigate whether or not there were differences between the HIE group and the healthy group in terms of NIRS parameters. The subjects were all term neonates, their age ranging from 2 to 18 days. The healthy group included 25 subjects while the HIE group consisted of 16 patients. A prototype NIRS instrument, which provides the data of tissue oxygenation including regional oxygen saturation (rSO2), the increment of oxyhemoglobin concentration and hemoglobin (deltaHbO2 and deltaHb) was used, and the data of rSO2 was compared with the data from the blood gas analyzer. The result shows that: (1) the mean+/-SD of rSO2 for the healthy group was 62 +/- 4% in the frontal region under the quiet sleep condition, but the mean+/-SD of rSO2 for the HIE group was 53 +/- 3%. (2) As all subjects inhaled pure oxygen in 21 min(-1) for a period of 60 s, rSO2 for the healthy group increased rapidly, with the increase in rSO2 (deltarSO2) being 7 +/- 2.3%, but the increase in rSO2 for the HIE neonates was 3 +/- 1.5%. After inhaling oxygen, deltaHbO2 and deltaHb between the two groups were also significantly different. (3) During all the experiments SpO2 was monitored, the value of SpO2 was not significantly different between the two groups. The above observations suggest that the rSO2 in quiet condition and the values of change of rSO2, HbO2 and Hb during the inhalation of oxygen may be used as the parameters to discover and assess the HIE infants.

A comparative assessment of the efficacy and side-effect liability of neuroprotective compounds in experimental stroke.

There are many examples of compounds showing neuroprotective efficacy in animal models of stroke but not in clinical trials. It is possible that some or all of these compounds possess poor therapeutic ratios, which results in the administration of sub-efficacious doses in order to avoid the emergence of side-effects. In order to explore this possibility, this study compared the therapeutic ratios of a number of neuroprotective agents that have undergone clinical trials. Neuroprotective efficacy was established using the mouse permanent (24 h) middle cerebral artery occlusion model. Side-effect liability was determined by assessment of motor coordination using the rotarod test. The therapeutic ratio was calculated as the ratio between the minimum effective dose (MED) for significant impairment in rotarod performance and the MED for significant neuroprotection. Compounds were administered i.p. 30 min prior to rotarod testing or onset of ischemia. Drugs such as Ifenprodil, Cerestat and Selfotel, that have failed in clinical trials, were found to have very low therapeutic ratios of < or = 1, whereas compounds with more tolerable clinical side-effect profiles were found to have higher therapeutic ratios (2, 10 and 10 for Sipatrigine, Remacemide and sPBN, respectively). It is concluded that the lack of efficacy of a number of neuroprotectants in clinical trials may well be a consequence of their poor therapeutic ratios.