Jane Hassell

Impact of anaesthetics and surgery on neurodevelopment: an update

Accumulating preclinical and clinical evidence suggests the possibility of neurotoxicity from neonatal exposure to general anaesthetics. Here, we review the weight of the evidence from both human and animal studies and discuss the putative mechanisms of injury and options for protective strategies. Our review identified 55 rodent studies, seven primate studies, and nine clinical studies of interest. While the preclinical data consistently demonstrate robust apoptosis in the nervous system after anaesthetic exposure, only a few studies have performed cognitive follow-up. Nonetheless, the emerging evidence that the primate brain is vulnerable to anaesthetic-induced apoptosis is of concern. The impact of surgery on anaesthetic-induced brain injury has not been adequately addressed yet. The clinical data, comprising largely retrospective cohort database analyses, are inconclusive, in part due to confounding variables inherent in these observational epidemiological approaches. This places even greater emphasis on prospective approaches to this problem, such as the ongoing GAS trial and PANDA study.

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.

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.

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 2–26 h 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 < 40 h were randomized to: (i) Cooling (33 °C) from 2–26 h (n = 10); or (ii) Cooling and inhaled 45–50% Argon (Cooling + Argon) from 2–26 h (n = 8). Whole-brain phosphorus-31 and regional proton MRS were acquired at baseline, 24 and 48 h after hypoxia–ischemia. EEG was monitored. At 48 h 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 48 h after hypoxia–ischemia (p < 0.001 for both) and lower 1H MRS lactate/N acetyl aspartate in white (p = 0.03 and 0.04) but not gray matter at 24 and 48 h. 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 mm2 were 23.9 points lower (95% C.I. 7.3–40.5) for the Cooling + Argon versus Cooling. Inhaled 45–50% Argon from 2–26 h augmented hypothermic protection at 48 h 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.

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.

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.

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.

Contribution of perinatal conditions to cerebral palsy in Uganda

Worldwide, an estimated 93 million children are disabled, 80% of whom live in low-income countries. Cohort studies in high-income countries attribute more than 50% of cerebral palsy cases to premature birth or prenatal causes. Equivalent data have been difficult to obtain in low-resource settings, limiting the ability to lobby for better service provision for these children. In October, 2017, Angelina KakoozaMwesige and colleagues published the largest population-based study of cerebral palsy in sub-Saharan Africa. This important work highlighted the higher prevalence of cerebral palsy in children in sub-Saharan Africa than in children in high-income countries, and that a greater proportion of cases are from potentially preventable causes. However, for diagnostic precision, consistent with all major international cerebral palsy registers, children younger than 2 years were not included. We suggest that further exploration of this younger group is important given the high proportion of full-term neonatal cases with severe cerebral palsy identified in this setting. We wish to add our experience of this important younger subgroup in an urban hospital setting in Kampala, Uganda. We investigated the causes and subtypes of cerebral palsy in 130 children younger than 18 years who presented to Mulago National Referral Hospital—the largest paediatric centre in Uganda—over an 8 week period in 2013. Children younger than 2 years were not excluded because of concern that those with severe cerebral palsy might not survive beyond early childhood. Children in whom there was concern about movement or posture were identified by doctors and therapists working in the paediatric in-patient wards and neurology, physiotherapy, and occupational therapy clinics. Children were recruited and assessed by the study lead (JH) or a local paediatrician trained in cerebral palsy assessment, assisted by a local language (Luganda) translator. Written consent was obtained. Cerebral palsy diagnosis was confirmed in cases of onset of non-progressive movement disorder before age 2 years. Children with neural tube defects or isolated hypotonia were excluded. Assessment involved obtaining detailed retrospective histories from primary caregivers, including any self-identified antecedents to the onset of motor impairment, and neurological examination. Cerebral palsy subtype was assigned according to the Surveillance of Cerebral Palsy in Europe hierarchical classification. Neuroimaging was not available. In this hospital-based cohort, 56% of patients were male, 9% were in-patients, and 63% were younger than 2 years (median age 17 months [IQR 9–29]). 78% of patients had bilateral spastic disease; 72% had four-limb spastic disease resulting in severe functional impairment. 68% of caregivers gave histories consistent with term intrapartumrelated encephalopathy or neonatal sepsis, and 76% attributed their child’s cerebral palsy to severe (full-term) neonatal illness, supported by a history of admission to neonatal care facilities (table). Two children were HIV positive. Key differences exist between our urban hospital-based cohort and the predominantly rural population-based cohort reported by Kakooza-Mwesige and colleagues. Our cohort was younger (median age 17 months vs 4–5 years), with more cases of fourlimb cerebral palsy, suggesting a higher prevalence of global brain injury that usually results from intrapartum or neonatal complications, as opposed to less disabling unilateral lesions. The younger age of patients and greater severity of disease seen in our hospital cohort might reflect increased care seeking or referral for these patients, as well as an increased incidence of comorbidities (eg, infection, malnutrition) precipitating

Optimum therapeutic hypothermia temperature after perinatal asphyxia: a magnetic resonance spectroscopy biomarker and immunohistochemistry study in the newborn piglet

Abstract Background Therapeutic hypothermia for neonatal encephalopathy improves survival with normal neurological outcome. Despite treatment, however, 50% of infants have adverse outcomes; customising cooling with more precision might provide further benefit. The specific brain temperature for optimum neuroprotection is unknown. We have previously shown in the piglet that optimum neuroprotection by delayed cooling occurs at different temperatures in the cortical (33°C) and deep grey (35°C) matter. Magnetic resonance biomarkers, including lactate:creatine ratio (Lac/Cr), predict neurodevelopmental outcome after neonatal encephalopathy. We aimed to assess the optimum temperature for regional neuroprotection in a proton magnetic resonance system (1H MRS). Methods 28 male piglets aged less than 24 h were anaesthetised with isoflurane and underwent trachaeal intubation, ventilation, continuous physiological monitoring, and intensive care treatment to maintain sedation and physiological homoeostasis. Within the bore of our 9·4 T magnetic resonance system all piglets underwent a hypoxic-ischaemic insult for 12·5 min, measured with real-time phosphorous MRS, before resuscitation. After this insult piglets were randomised (seven in each group) to normothermia (38·5°C), hypothermia (35°C), hypothermia (33·5°C), and hypothermia (30°C). Serial MRS assessments were acquired before, during, and up to 48 h after transient cerebral hypoxia-ischaemia. Areas under the curve (AUC) for the 1H MRS Lac/Cr peak-area ratio in ventromedial forebrain (vmFB, predominantly grey matter) and dorsal subcortical (dsc, predominantly white matter) voxels were calculated. Programmed cell death at 48 h was measured with terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining on paraffin-embedded tissue in corresponding regions. Findings Compared with normothermia, cooling to 35°C and 33·5°C produced consistent 50–75% reduction in density of TUNEL-positive cells with significant decreases in insular cortex, hippocampus, subcortical white matter, thalamus, and putamen (p Interpretation Cooling to 30°C worsened hypoxic-ischaemic injury in the thalamus and basal ganglia compared with cooling to 33·5°C and 35°C. We showed a higher threshold optimum temperature for neuroprotection in the deep grey matter than in white matter/dCTX on the basis of immunohistochemical markers of cell death. Systemic effects of cooling to 30°C might have exacerbated these detrimental effects on deep grey matter cell death. These data support previous work in the developing brain and are relevant to clinical practice for optimum therapeutic hypothermia in newborn infants with birth asphyxia. Funding UK Medical Research Council, National Institute for Health Research.

Investigation of cerebral autoregulation in the newborn piglet during anaesthesia and surgery.

The relationship between cerebral autoregulation (CA) and the neurotoxic effects of anaesthesia with and without surgery is investigated. Newborn piglets were randomly assigned to receive either 6 h of anaesthesia (isoflurane) or the same with an additional hour of minor surgery. The effect of the spontaneous changes in mean arterial blood pressure (MABP) on the cerebral haemodynamics (oxy- and deoxy-haemoglobin, HbO2 and Hb) was measured using transverse broadband near-infrared spectroscopy (NIRS). A marker for impaired CA, concordance between MABP and intravascular oxygenation (HbD = HbO2 − Hb) in the ultra-low frequency domain (0.0018–0.0083 Hz), was assessed using coherence analysis. Presence of CA impairment was not significant but found to increase with surgical exacerbation. The impairment did not correlate with histological outcome (presence of cell death, apoptosis and microglial activation in the brain).