Treatment advances in neonatal neuroprotection and neurointensive care

Abstract

Knowledge of the nature, prognosis, and ways to treat brain lesions in neonatal infants has increased remarkably. Neonatal hypoxic-ischaemic encephalopathy (HIE) in term infants, mirrors a progressive cascade of excito-oxidative events that unfold in the brain after an asphyxial insult. In the laboratory, this cascade can be blocked to protect brain tissue through the process of neuroprotection. However, proof of a clinical effect was lacking until the publication of three positive randomised controlled trials of moderate hypothermia for term infants with HIE. These results have greatly improved treatment prospects for babies with asphyxia and altered understanding of the theory of neuroprotection. The studies show that moderate hypothermia within 6 h of asphyxia improves survival without cerebral palsy or other disability by about 40% and reduces death or neurological disability by nearly 30%. The search is on to discover adjuvant treatments that can further enhance the effects of hypothermia.

Figure 1. Excito-oxidative cascade of events that mediate hypoxic-ischaemic brain injury

Severe hypoxia impairs oxidative metabolism leading to neuronal depolarisation and ischaemia. Ischaemia reduces delivery of glucose necessary for anaerobic metabolism, which powers neurotransmitter reuptake pumps on perisynaptic astrocytes.This leads to flooding of the synaptic cleft with glutamate and neuronal depolarisation, which in turn trigger opening of NMDA receptor channels and other calcium channels including acid-sensing ion channels, leading to excess calcium influx into neurons. Calcium flooding through NMDA channels activates the enzyme nitric oxide synthetase leading to high levels of the toxic free radical neurotransmitter nitric oxide. This toxic free radical, along with additional oxygen free radicals generated by reoxygenation of mitochondria following a period of hypoxia, attack enzymes associated with oxidative phosphorylation and electron transport. Calcium toxicity is also mediated by activation of other enzymes including caspases, calpains, other proteases, and lipases that attack mitochondria and other cellular machinery. Signals released from damaged mitochondria lead to apoptosis or programmed cell death as long as energy supplies persist, but exhaustion of energy supplies leads to necrosis in which cellular membranes are destroyed. Lactic acid accumulates when oxidative phosphorylation within mitochondria is impaired, but its toxicity seems to be less important in the neonatal brain than in adults. Similarly, cerebral oedema occurs when pumps required for water homoeostasis are impaired by reduced energy supplies owing to damaged mitochondria. Oedema seems to be a sign of energy failure rather than causing damage on its own. This excito-oxidative cascade occurs over a period of days to weeks. EAAT=excitatory aminoacid transporter. Gln=glutamine. Glu=glutamate. nNOS=neuronal nitric oxide synthase. NO=nitric oxide. VDCC=voltage-dependent calcium channels.

Figure 2. Downstream signalling pathways that mediate the apoptosis–necrosis continuum

Delayed cell death signalling pathways mediate the effects of hypoxia-ischaemia in the brain. The extrinsic pathway mirrors the cells’ external environment and begins when inflammatory cytokines (to the right of the figure at the cell surface) bind to and activate Fas-cell death receptors, whereas the intrinisic pathway is activated when signals released from within stressed mitochondria activate caspase and non-caspase-mediated-cell-death pathways within the nucleus. These two cell-death pathways activate common signalling networks within the mitochondria and the nucleus. Mitochondria exposed to caspase-induced stress can release cytochrome C through channels formed by the Bax and Bak proteins in the outer mitochondrial membrane (caspase-mediated cell death) or can release apoptosis inducing factor (AIF), which activates DNA fragmentation directly (non-caspase pathway). Cytochrome C can combine with Apaf1 and caspase 9 to form the apoptosome, which triggers activation of caspase 3. DNA breaks mediated by free radicals such as nitric oxide (NO·) and peroxynitrite activate poly-ADP-ribose polymerase 1 (PARP1), which consumes NAD+ and worsens the energy shortage for mitochondria (shown within the nucleus). Convergence of signalling for the extrinsic (Fas) and intrinsic cell-death pathways are responsible for the interaction between infection (endotoxin) and hypoxia-ischaemia that increases cell death. Vm=membrane potential. VSSC=voltage sensitive calcium channel. Fas=death receptor in tumour necrosis factor family. FADD=fas adaptor death domain protein. BclXL=antiapoptotic proteins in the Bcl2 family of proteins. Bax and Bak=proapoptotic proteins that form channels in outer mitochondrial membrane releasing cytochorome C to trigger apoptosis. tBid=truncated BH3-only proapoptotic protein. Apaf1=apoptotic protein activating factor 1. SMAC=antagonist of inhibitor of apoptosis. IAP=inhibitor of apoptosis. CAD=caspase activating DNAse. Cyt C=cytochrome C. nNOS=neuronal nitric oxide synthase. NO=nitric oxide. ROS=reactive oxygen species. PARP1=poly-ADP-ribose polymerase 1. PAR=poly-ADP ribose formed by ribosylation of DNA and proteins. GSH=glutathione, an antioxidant. AIF=apoptosis-inducing factor. Bid=BH3 interacting domain proapoptotic protein. NAD=nicotinamide adenine dinucleotide. FASL=Fas death receptor ligand.

Figure 3. Delayed apoptosis in a Vannucci model of unilateral hypoxia-ischaemia in 7-day-old rat pups

This neonatal model of brain injury shows that neurons continue to commit to programmed cell death over several days after injury. (A) The top diagram shows the distribution of neurons in coronal slices of brain tissue that express caspase 3, a marker for apoptosis, at intervals up to 168 h after injury. (B) This graph shows the delayed appearance of necrotic or apoptotic cells in the same coronal sections of brain tissue shown in A, using a standard histopathological scoring system. The Y-axis shows the abundance of these dying cells in the cerebral cortex, corpus striatum, or the CA1 region of the hippocampus. (C) Electron microscopy images (magnification ×2500) of apoptotic neurons at different stages of programmed cell death from the thalamus (two on the left) and hippocampus (on the right) showing characteristic dark, condensed chromatin. Early apoptotic cells show some nuclear condensation, whereas cells in more advanced stages of cell death show extensive nuclear and cytoplasmic condensation.

Figure 4. T1-weighted MRI of a baby at 2 weeks of age who had undergone severe, near-total asphyxia around the time of birth

Asphyxia was associated with a cord blood pH of 7·6 and base deficit of 25 mEg/L (calculated base deficit in standard arterial blood gases). Injured T1-enhancing areas are very focal and localised to regions of the thalamus, putamen, and peri-Rolandic cerebral cortex that contain synapses that connect the developing motor circuits.