Giles S. Kendall

Two sodium channels contribute to the TTX-R sodium current in primary sensory neurons

We have cloned a second tetrodotoxin-resistant (TTX-R) sodium channel α subunit, SNS2, with the same amino-acid sequence as a putative sodium channel α subunit NaN (ref. 1). SNS2 expression in HEK293T cells produced a TTX-R voltage-gated sodium current with faster kinetics and a lower TTX IC50 than the previously cloned sensory-neuron-specific sodium channel, SNS/PN3 (Refs 2, 3). SNS2 was co-expressed with SNS/PN3 in small DRG neurons.

Passive cooling for initiation of therapeutic hypothermia in neonatal encephalopathy

Objective To determine the feasibility of passive cooling to initiate therapeutic hypothermia before and during transport. Methods Consensus guidelines were developed for passive cooling at the referring hospital and on transport by the London Neonatal Transfer Service. These were evaluated in a prospective study. Results Between January and October 2009, 39 infants were referred for therapeutic hypothermia; passive cooling was initiated at the referring hospital in all the cases. Despite guidance, no rectal temperature measurements were taken before arrival of the transfer team. Cooling below target temperature (33°C–34°C) occurred in five babies before the arrival of the transfer team. In two of these infants, active cooling was performed, rectal temperature was not recorded and their temperature was lower than 32°C. Of the remaining 37 babies, 33 (89%) demonstrated a reduction in core temperature with passive cooling alone. The percentage of the babies within the temperature range at referral, arrival of the transfer team and arrival at the cooling centre were 0%, 15% and 67%, respectively. On arrival at the cooling centre, four babies had cooled to lower than 33°C by passive cooling alone (32.7°C, 32.6°C, 32.2°C and 32.1°C). Initiation of passive cooling before and during transfer resulted in the therapy starting 4.6 (1.8) h earlier than if initiated on arrival at the cooling centre. Conclusions Passive cooling is a simple and effective technique if portable cooling equipment is unavailable. Rectal temperature monitoring is essential; active cooling methods without core temperature monitoring may lead to overcooling.

AdaPT: An adaptive preterm segmentation algorithm for neonatal brain MRI

Advances in neonatal care have improved the survival of infants born prematurely although these infants remain at increased risk of adverse neurodevelopmental outcome. The measurement of white matter structure and features of the cortical surface can help define biomarkers that predict this risk. The measurement of these structures relies upon accurate automated segmentation routines, but these are often confounded by neonatal-specific imaging difficulties including poor contrast, low resolution, partial volume effects and the presence of significant natural and pathological anatomical variability. In this work we develop and evaluate an adaptive preterm multi-modal maximum a posteriori expectation-maximisation segmentation algorithm (AdaPT) incorporating an iterative relaxation strategy that adapts the tissue proportion priors toward the subject data. Also incorporated are intensity non-uniformity correction, a spatial homogeneity term in the form of a Markov random field and furthermore, the proposed method explicitly models the partial volume effect specifically mitigating the neonatal specific grey and white matter contrast inversion. Spatial priors are iteratively relaxed, enabling the segmentation of images with high anatomical disparity from a normal population. Experiments performed on a clinical cohort of 92 infants are validated against manual segmentation of normal and pathological cortical grey matter, cerebellum and ventricular volumes. Dice overlap scores increase significantly when compared to a widely-used maximum likelihood expectation maximisation algorithm for pathological cortical grey matter, cerebellum and ventricular volumes. Adaptive maximum a posteriori expectation maximisation is shown to be a useful tool for accurate and robust neonatal brain segmentation.

TNF gene cluster deletion abolishes lipopolysaccharide-mediated sensitization of the neonatal brain to hypoxic ischemic insult

In the current study, we explored the role of TNF cluster cytokines on the lipopolysaccharide (LPS)-mediated, synergistic increase in brain injury after hypoxic ischemic insult in postnatal day 7 mice. Pretreatment with moderate doses of LPS (0.3 μg/g) resulted in particularly pronounced synergistic injury within 12 h. Systemic application of LPS alone resulted in a strong upregulation of inflammation-associated cytokines TNFα, LTβ, interleukin (IL) 1β, IL6, chemokines, such as CXCL1, and adhesion molecules E-Selectin, P-Selectin and intercellular adhesion molecule-1 (ICAM1), as well as a trend toward increased LTα levels in day 7 mouse forebrain. In addition, it was also associated with strong activation of brain blood vessel endothelia and local microglial cells. Here, deletion of the entire TNF gene cluster, removing TNFα, LTβ and LTα completely abolished endotoxin-mediated increase in the volume of cerebral infarct. Interestingly, the same deletion also prevented endothelial and microglial activation following application of LPS alone, suggesting the involvement of these cell types in bringing about the LPS-mediated sensitization to neonatal brain injury.

Techniques for therapeutic hypothermia during transport and in hospital for perinatal asphyxial encephalopathy

Over the past 10 years, several randomised clinical trials of therapeutic hypothermia for perinatal asphyxial encephalopathy have demonstrated both safety and efficacy of therapeutic hypothermia in improving neurological outcome. Today cooling is increasingly used in tertiary level units throughout the developed world. Therapeutic hypothermia (cooling to a rectal or core temperature of 33-34 degrees C for 72 h) is easier to achieve in newborn infants than in adults. There is a natural tendency for the core temperature of infants who suffered birth asphyxia to fall and remain lower than non-asphyxiated infants for up to 16 h after birth. A variety of high- and low-tech surface cooling methods have been used in neonates - newer systems are servo-controlled and provide very stable temperature control. It is well accepted that to be most effective, cooling needs to be initiated as soon as possible after birth and, thus, needs to be commenced prior to the transfer of infants to cooling centres. We describe our experience of passive cooling before and during the transfer of infants with encephalopathy to cooling centres in a major city in the UK.

N-methyl-isobutyl-amiloride ameliorates brain injury when commenced before hypoxia ischemia in neonatal mice

Underphysiologic conditions, brain intracellular pH (pHi) is maintained at 7.03. Rebound brain intracellular alkalosis has been observed in experimental models and adult stroke after hypoxia/ischemia (HI). In term infants with neonatal encephalopathy (NE), an association exists between the magnitude of brain alkalosis and neurodevelopmental outcome, and there is increasing evidence to suggest that alkalosis may be deleterious to cell survival. Activation of the Na+/H+ exchanger (NHE) is thought to be responsible for the rapid normalization of pHi and rebound alkalosis after reperfusion. We hypothesized that N-methyl-isobutyl-amiloride (MIA), an inhibitor of the NHE, would reduce brain injury in a model of neonatal HI. Seven-day-old mice underwent left carotid artery occlusion followed by exposure to 8% oxygen for 30 min (moderate insult) or 1 h (severe insult). Animals received MIA or saline 8 hourly starting 30 min before HI. Outcome was determined at 48 h by measuring viable tissue in the injured hemisphere (severe insult) or injury score and TUNEL staining (moderate insult). After the severe insult, MIA had a significant neuroprotective effect increasing forebrain tissue survival from 44% to 67%. After the moderate insult, damage was localized to the hippocampus where treatment resulted in a significant reduction in injury score and in TUNEL-positive cells. MIA was also shown to have a significant overall neuroprotective effect based on injury score after the moderate insult. Amiloride analogues are neuroprotective when commenced before HI in a mouse model.

A MINIMAL CGRP GENE PROMOTER IS INDUCIBLE BY NERVE GROWTH FACTOR IN ADULT RAT DORSAL ROOT GANGLION NEURONS BUT NOT IN PC12 PHAEOCHROMOCYTOMA CELLS

The calcitonin/CGRP gene is transcribed in thyroid C cells and some neuronal cells but not in other cell types. Although the promoter sequences mediating gene activity in thyroid C cells have been extensively studied, the elements responsible for promoter activity in neuronal cells and its stimulation by nerve growth factor (NGF) have not previously been defined. We report the first use of the calcium phosphate procedure to successfully transfect adult rat dorsal root ganglion neurons, which naturally express the calcitonin/calcitonin gene‐related peptide (CGRP) in an NGF‐inducible manner. This method was used to characterize the elements in the calcitonin/CGRP promoter which are responsible for its basal activity and NGF inducibility in DRG neurons and in PC12 cells, a neuronally derived cell line which does not naturally express the calcitonin/CGRP gene. Although the sequences required for basal activity are similar in each cell type, we show that a minimal calcitonin/CGRP promoter is NGF‐responsive in dorsal root ganglion cells, but that upstream sequences are required for such inducibility in PC12 cells.

Nerve growth factor induces expression of immediate-early genes NGFI-A (Egr-1) and NGFI-B (nur 77) in adult rat dorsal root ganglion neurons

We have used primary cultures of adult rat dorsal root ganglia (DRG), enriched in sensory neurons, to investigate the induction of immediate-early genes by NGF and a variety of other growth factors. Using the polymerase chain reaction we have quantitatively amplified specific mRNA transcripts induced by NGF, in the presence and absence of the protein synthesis inhibitor cycloheximide. NGFIA (Egr-1) and NGFIB (nur 77) mRNAs were elevated in level within 60 min of NGF treatment and independently of de novo protein synthesis. This was consistent with the behaviour of immediate-early genes. These kinetics were seen at a range of NGF concentrations. NGFIA and NGFIB mRNAs were also found to be induced in DRG cultures by a variety of other growth factors. Different patterns of induction of NGFIA and NGFIB mRNA observed in DRG cultures suggested that transcript-specific pathways of signal transduction were operating within neurons, dependent upon the particular growth factor stimulus. Comparison of data reported from growth factor treatment of other cell types with data from DRG cultures also revealed patterns of NGFIA and NGFIB mRNA induction specific to DRG neurons.

Preterm birth affects the developmental synergy between cortical folding and cortical connectivity observed on multimodal MRI.

The survival rates of infants born prematurely have improved as a result of advances in neonatal care, although there remains an increased risk of subsequent disability. Accurate measurement of the shape and appearance of the very preterm brain at term-equivalent age may guide the development of predictive biomarkers of neurological outcome. We demonstrate in 92 preterm infants (born at an average gestational age of 27.0±2.7weeks) scanned at term equivalent age (scanned at 40.4±1.74weeks) that the cortical sulcation ratio varies spatially over the cortical surface at term equivalent age and correlates significantly with gestational age at birth (r=0.49,p<0.0001). In the underlying white matter, fractional anisotropy of local white matter regions correlated significantly with gestational age at birth at term equivalent age (for the genu of the corpus callosum r=0.26,p=0.02 and for the splenium r=0.52,p<0.001) and in addition the fractional anisotropy in these local regions varies according to location. Finally, we demonstrate that connectivity measurements from tractography correlate significantly and specifically with the sulcation ratio of the overlying cortical surface at term equivalent age in a subgroup of 20 infants (r={0.67,0.61,0.86}, p={0.004,0.01,0.00002}) for tract systems emanating from the left and right corticospinal tracts and the corpus callosum respectively). Combined, these results suggest a close relationship between the cortical surface phenotype and underlying white matter structure assessed by diffusion weighted MRI. The spatial surface pattern may allow inference on the connectivity and developmental trajectory of the underlying white matter complementary to diffusion imaging and this result may guide the development of biomarkers of functional outcome.

Longitudinal measurement of the developing grey matter in preterm subjects using multi-modal MRI.

Preterm birth is a major public health concern, with the severity and occurrence of adverse outcome increasing with earlier delivery. Being born preterm disrupts a time of rapid brain development: in addition to volumetric growth, the cortex folds, myelination is occurring and there are changes on the cellular level. These neurological events have been imaged non-invasively using diffusion-weighted (DW) MRI. In this population, there has been a focus on examining diffusion in the white matter, but the grey matter is also critically important for neurological health. We acquired multi-shell high-resolution diffusion data on 12 infants born at ≤ 28 weeks of gestational age at two time-points: once when stable after birth, and again at term-equivalent age. We used the Neurite Orientation Dispersion and Density Imaging model (NODDI) (Zhang et al., 2012) to analyse the changes in the cerebral cortex and the thalamus, both grey matter regions. We showed region-dependent changes in NODDI parameters over the preterm period, highlighting underlying changes specific to the microstructure. This work is the first time that NODDI parameters have been evaluated in both the cortical and the thalamic grey matter as a function of age in preterm infants, offering a unique insight into neuro-development in this at-risk population.