Whole-Genome Sequencing of Retinoblastoma Reveals the Diversity of Rearrangements Disrupting RB1 and Uncovers a Treatment-Related Mutational Signature.

The development of retinoblastoma is thought to require pathological genetic changes in both alleles of the RB1 gene. However, cases exist where RB1 mutations are undetectable, suggesting alternative pathways to malignancy. We used whole-genome sequencing (WGS) and transcriptomics to investigate the landscape of sporadic retinoblastomas derived from twenty patients, sought RB1 and other driver mutations and investigated mutational signatures. At least one RB1 mutation was identified in all retinoblastomas, including new mutations in addition to those previously identified by clinical screening. Ten tumours carried structural rearrangements involving RB1 ranging from relatively simple to extremely complex rearrangement patterns, including a chromothripsis-like pattern in one tumour. Bilateral tumours obtained from one patient harboured conserved germline but divergent somatic RB1 mutations, indicating independent evolution. Mutational signature analysis showed predominance of signatures associated with cell division, an absence of ultraviolet-related DNA damage and a profound platinum-related mutational signature in a chemotherapy-exposed tumour. Most RB1 mutations are identifiable by clinical screening. However, the increased resolution and ability to detect otherwise elusive rearrangements by WGS have important repercussions on clinical management and advice on recurrence risks.

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.

Systemic pro-inflammatory cytokine status following therapeutic hypothermia in a piglet hypoxia-ischemia model.

BACKGROUND: Inflammatory cytokines are implicated in the pathogenesis of perinatal hypoxia-ischemia (HI). The influence of hypothermia (HT) on cytokines after HI is unclear. Our aim was to assess in a piglet asphyxia model, under normothermic (NT) and HT conditions: (i) the evolution of serum cytokines over 48 h and (ii) cerebrospinal fluid (CSF) cytokine levels at 48 h; (iii) serum pro/anti-inflammatory cytokine profile over 48 h and (iv) relation between brain injury measured by magnetic resonance spectroscopy (MRS) and brain TUNEL positive cells with serum cytokines, serum pro/anti-inflammatory cytokines and CSF cytokines. METHODS: Newborn piglets were randomized to NT (n = 5) or HT (n = 6) lasting 2-26 h after HI. Serum samples were obtained 4-6 h before, during and at 6-12 h intervals after HI; CSF was obtained at 48 h. Concentrations of interleukin (IL)-1ß, -4, -6, -8, -10 and TNF-α were measured and pro/anti-inflammatory status compared between groups. White matter and thalamic voxel lactate/N-acetyl aspartate (Lac/NAA) (a measure of both oxidative metabolism and neuronal loss) were acquired at baseline, after HI and at 24 and 36 h. RESULTS: Lac/NAA was reduced at 36 h with HT compared to NT (p = 0.013 basal ganglia and p = 0.033 white matter). HT showed lower serum TNF-α from baseline to 12 h (p < 0.05). Time-matched (acquired within 5 h of each other) serum cytokine and MRS showed correlations between Lac/NAA and serum IL-1ß and IL-10 (all p < 0.01). The pro/anti-inflammatory ratios IL-1ß/IL-10, IL-6/IL-10, IL-4/IL-10 and IL-8/IL-10 were similar in NT and HT groups until 36 h (24 h for IL-6/IL-10); after this, 36 h pro/anti-inflammatory cytokine ratios in the serum were higher in HT compared to NT (p < 0.05), indicating a pro-inflammatory cytokine surge after rewarming in the HT group. In the CSF at 48 h, IL-8 was lower in the HT group (p < 0.05). At 48 h, CSF TNF-α correlated with Lac/NAA (p = 0.02) and CSF IL-8 correlated with white matter TUNEL positive cell death (p = 0.04). CONCLUSIONS: Following cerebral HI, there was a systemic pro-inflammatory surge after rewarming in the HT group, which is counterintuitive to the putative neuroprotective effects of HT. While serum cytokines were variable, elevations in CSF inflammatory cytokines at 48 h were associated with MRS Lac/NAA and white matter cell death.

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.

Placental, Matrilineal, and Epigenetic Mechanisms Promoting Environmentally Adaptive Development of the Mammalian Brain.

The evolution of intrauterine development, vivipary, and placentation in eutherian mammals has introduced new possibilities and constraints in the regulation of neural plasticity and development which promote neural function that is adaptive to the environment that a developing brain is likely to encounter in the future. A range of evolutionary adaptations associated with placentation transfers disproportionate control of this process to the matriline, a period unique in mammalian development in that there are three matrilineal genomes interacting in the same organism at the same time (maternal, foetal, and postmeiotic oocytes). The interactions between the maternal and developing foetal hypothalamus and placenta can provide a template by which a mother can transmit potentially adaptive information concerning potential future environmental conditions to the developing brain. In conjunction with genomic imprinting, it also provides a template to integrate epigenetic information from both maternal and paternal lineages. Placentation also hands ultimate control of genomic imprinting and intergenerational epigenetic information transfer to the matriline as epigenetic markers undergo erasure and reprogramming in the developing oocyte. These developments, in conjunction with an expanded neocortex, provide a unique evolutionary template by which matrilineal transfer of maternal care, resources, and culture can be used to promote brain development and infant survival.

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.

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.

The post-natal chemosensory environment induces epigenetic changes in vomeronasal receptor gene expression and a bias in olfactory preference.

Vomeronasal stem cells are generated throughout the life of a mouse and differentiate into neurons that express one vomeronasal type 1 (V1r), one or two vomeronasal type 2 (V2r), or one olfactory receptor. Vomeronasal stem cells can be induced to differentiate into neurons by treatment with lipocalins from mouse urine or by epigenetic modification following treatment with histone deacetylase inhibitors. An important question is, do chemosensory signals, modify the detection capabilities of the vomeronasal organ and affect behaviour. Rearing mice in the presence of urine (and its pheromonal signals) derived from a different mouse strain, affected the behavioural preference for non-kin which were accompanied by changes in vomeronasal receptor expression. Significant changes in the expression of vomeronasal V1r, V2r and olfactory receptors, major urinary proteins, and a number of genes thought to be involved in transcriptional regulation were also observed following urine treatment. These results suggest that modification of a mouse's urinary environment may exert epigenetic effects on developing vomeronasal neurons, which modify the type of vomeronasal receptors that are expressed. This may provide a mechanism by which environmental changes are able to modify the detection capabilities of the vomeronasal organ to respond optimally to the most likely social environment that a mouse will encounter when mature.

Placental protection of the fetal brain during short-term food deprivation.

The fetal genome regulates maternal physiology and behavior via its placenta, which produces hormones that act on the maternal hypothalamus. At the same time, the fetus itself develops a hypothalamus. In this study we show that many of the genes that regulate placental development also regulate the developing hypothalamus, and in mouse the coexpression of these genes is particularly high on embryonic days 12 and 13 (days E12-13). Such synchronized expression is regulated, in part, by the maternally imprinted gene, paternally expressed gene 3 (Peg3), which also is developmentally coexpressed in the hypothalamus and placenta at days E12-13. We further show that challenging this genomic linkage of hypothalamus and placenta with 24-h food deprivation results in disruption to coexpressed genes, primarily by affecting placental gene expression. Food deprivation also produces a significant decrease in Peg3 gene expression in the placenta, with consequences similar to many of the placental gene changes induced by Peg3 mutation. Such genomic dysregulation does not occur in the hypothalamus, where Peg3 expression increases with food deprivation. Thus, changes in gene expression brought about by food deprivation are consistent with the fetal genome's maintaining hypothalamic development at a cost to its placenta. This biased change to gene dysregulation in the placenta is linked to autophagy and ribosomal turnover, which sustain, in the short term, nutrient supply for the developing hypothalamus. Thus, the fetus controls its own destiny in times of acute starvation by short-term sacrifice of the placenta to preserve brain development.

Increased apoptosis during neonatal brain development underlies the adult behavioral deficits seen in mice lacking a functional paternally expressed gene 3 (Peg3).

Inactivation of the maternally imprinted, paternally expressed gene 3 (Peg3) induces deficits in olfactory function, sexual and maternal behaviors, oxytocin neuron number, metabolic homeostasis and growth. Peg3 is expressed in a number of developing hypothalamic and basal forebrain structures and is a component of the P53 apoptosis pathway. Peg3 inactivation in neuronal cell culture lines inhibits P53 mediated apoptosis, which is important in the early postnatal development and sexual differentiation of the brain. In this study, we investigated the effect of inactivating the Peg3 gene on the incidence of caspase 3 positive cells (a marker of apoptosis) in 4- and 6-day postpartum mouse brain. Inactivating the Peg3 gene resulted in an increase in the incidence of total forebrain caspase 3 positive cells at 4 and 6 days postpartum. Increases in specific neuroanatomical regions including the bed nucleus of the stria terminalis, nucleus accumbens, caudate putamen, medial pre-optic area, arcuate nucleus, medial amygdala, anterior cortical and posteriodorsal amygdaloid nuclei, were also observed. In wild-type mice, sex differences in the incidence of caspase 3 positive cells in the medial amygdala, bed nucleus of the stria terminalis, nucleus accumbens, arcuate nucleus and the M2 motor cortex, were also observed. This neural sex difference was ameliorated in the Peg-3 mutant. These findings suggest that the neuronal and behavioral deficits seen in mice lacking a functional Peg3 gene are mediated by increases in the incidence of early neonatal apoptosis in neuroanatomical regions important for reproductive behavior, olfactory and pheromonal processing, thermoregulation and reward.

Steroid control of gonadotropin-releasing hormone secretion: associated changes in pro-opiomelanocortin and preproenkephalin messenger RNA expression in the ovine hypothalamus.

The endogenous opioid peptides have been implicated in mediating the actions of estrogen and progesterone on GnRH release. We used in situ hybridization histochemistry to determine whether steroid-induced changes in GnRH/LH release in the female sheep are associated with changes in the cellular mRNA content of the precursors for beta-endorphin (pro-opiomelanocortin; POMC) and met-enkephalin (pre-proenkephalin; PENK). Two specific hypotheses were tested. First, that the inhibitory actions of progesterone are associated with an increase in opioid gene expression in specific hypothalamic nuclei. Our data support this hypothesis. Thus, an increase in progesterone was associated with increased POMC gene expression in the arcuate nucleus and PENK in the paraventricular nucleus. Further, the increase in POMC was restricted to regions of the arcuate nucleus that contain steroid sensitive beta-endorphin neurons. Our second hypothesis, that gene expression for the two opioid precursors would decrease prior to the start of the estradiol-stimulated GnRH surge, was not supported. Rather, POMC (but not PENK) gene expression in the arcuate nucleus was significantly higher in estradiol-treated animals than controls at the peak of the GnRH surge. These data suggest that beta-endorphin neurons in subdivisions of the arcuate nucleus and enkephalin neurons in the paraventricular nucleus are part of the neural network by which progesterone inhibits LH release. While enkephalin neurons may not play a role in estrogen positive feedback, increases in POMC mRNA in the arcuate nucleus at the time of the GnRH peak may be important for replenishing beta-endorphin stores and terminating estrous behavior.

Increased BDNF and trk-B mRNA expression in cortical and limbic regions following formation of a social recognition memory.

Brain-derived neurotrophic factor (BDNF) and its receptor, tyrosine receptor kinase (trk-B), play important roles in neural plasticity, long-term potentiation and memory formation. Sheep form a selective recognition memory for their lambs within 2 h of birth. Initially, this memory is exclusively based on olfactory cues; however, as it consolidates over a 12-h recognition period it extends to incorporate visual cues. We investigated whether changes in BDNF and trk-B mRNA expression occurred in both olfactory and visual processing systems at 4.5 h postpartum, 2-3 h after the behavioural manifestations of an olfactory recognition memory were found. Animals that formed a recognition memory showed increased BDNF mRNA expression in the inferior part of the temporal cortex, subfield CA1 of the hippocampus, the diagonal band, basolateral amygdala and the anterior cingulate, medial frontal, entorhinal and pyriform cortices. No increases were observed in either the olfactory bulbs or the dentate gyrus. Expression of trk-B mRNA was significantly increased only in the medial temporal, entorhinal and pyriform cortices. These findings demonstrate that by 2-3 h following the initial formation of olfactory recognition memory there are BDNF/trk-B-mediated plasticity changes in brain areas involved in the consolidation of olfactory memory (the pyriform and entorhinal cortices). However, similar changes also occur in areas of the brain involved in visual memory, face and object recognition (the temporal cortex, entorhinal cortex, hippocampal subfield CA1 and basolateral amygdala), and in areas of the brain with integrative and attentional functions (the medial frontal and anterior cingulate cortices and diagonal band). This suggests that reorganization of neural circuits underlying the visual recognition of lambs or the integration of olfactory/visual information is occurring even at this time even though accurate behavioural recognition at this stage can only be made using olfactory cues.