Cellular and Molecular Mechanisms of Neonatal Brain Injury and Neuroprotection

Acknowledgments

We thank Drs. Billie Short, Nickie Andescavage, and Nneka Nzegwu (Division of Neonatology, Children’s National Hospital); Dr. Taeun Chang (Neonatal Neurology Program, Division of Epilepsy, Neurophysiology & Critical Care, Children’s National Hospital); and Dr. Hideo Jinno (Center for Neuroscience Research, Children’s National Hospital and Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences) for critically reading this chapter. The research program of the authors of this chapter is supported by award numbers R37NS109478 (Javits Award) and R01NS105138 from NINDS (V.G.), R01NS099461 (J.S.) and by an award from the Board of Visitors of Children’s National Hospital (P.K.). The authors’ work is also supported by the District of Columbia Intellectual and Developmental Disabilities Research Center U54HD090257 (V.G.) from Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). This content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Introduction

Brain injury in neonates remains a leading cause of neurodevelopmental delays, long-term morbidity, and death. The effects of injury in the newborn brain are evident in domains related to motor function, social, behavioral, cognitive, and mental health. ^, While the long-term results of neonatal brain injury are clear, a new mechanistic understanding of the specific neural cell populations and regions vulnerable to injury is necessary in order to define rational therapies. Due to the in utero and ex utero dynamic state of the developing brain, the patterns of damage in different central nervous system (CNS) regions may vary significantly. The premature nervous system is vulnerable to injurious events that potentially alter its framework and functions. Early loss of placental support, hypoxia, emboli, substance exposure, inflammation, and hormonal dysregulation are some of the known predisposing factors to neonatal brain injury. ^, The susceptibility of the developing brain to injurious insults is multifactorial and depends on the anatomic location and the developmental event that is affected by the insult. Neurons and glial cells are also differentially susceptible to injury. Due to maturational changes (neurogenesis and neural circuit formation, glial cell differentiation and myelination) occurring in different developmental epochs, one could anticipate patterns of injury that correspond to specific neural cell types and the developmental trajectories to be affected.

Acute causes of neonatal brain injury in the neonatal intensive care unit (NICU) include hypoxia, ischemia, inflammation, hemorrhage, infection, metabolic disorders, and any combination of these events. Importantly, insults may occur during the prenatal period and have a role in initiating or regulating perinatal brain injury. Finally, different causes of brain injury, either in the term or preterm neonate, eventually converge into common cellular and molecular pathways that we will describe and discuss in this chapter.

Impact of Neonatal Brain Injury

Both term and preterm born neonates are susceptible to severe brain injury, although the classic causes vary. For term infants, neonatal hypoxic-ischemic encephalopathy (HIE), has an incidence of 1 to 8/1000 live births worldwide with a high risk of death (85% in severe HIE), cerebral palsy (CP), and developmental disabilities ( Fig. 133.1 ). ^, ^, Preterm birth, occurring in 1 in 10 infants in the United States (approximately 15 million of all births worldwide per year), and its associated complications are important causes of mortality in children under 5 years of age. Neurodevelopmental impairments include CP, learning disabilities, neuropsychiatric disorders, and potentially, autism spectrum disorder (ASD). ^, Several studies have documented a strong correlation between prematurity and deficits in auditory, visual, and tactile sensory domains. ^, The prevalence of neuropsychiatric diseases, including diagnosis of ASD, attention-deficit/hyperactivity disorder (ADHD), schizophrenia, anxiety, and behavioral disorders, is four to five times higher in children and adults who were born prematurely. ^, ^, Indeed, because very few long-term studies of preterm infants have been carried out, the behavioral and psychiatric impact may be underestimated. ^, In addition, the financial burden associated with premature birth in the United States, including the inability to work due to lack of independence or ability, is estimated at $26.2 billion each year.

Fig. 133.1, Diffusion-weighted magnetic resonance imaging (DW-MRI) shows neonatal stroke in a term infant. The infant displayed seizures at 16 hours of life. Patient exam revealed an encephalopathic infant with left sided hypotonia and hyporeflexia—arm greater than leg. Electroencephalogram (EEG) demonstrated attenuated signal on the right and multifocal seizures emanating from the right central-temporal region. MRI demonstrates large right middle cerebral artery ischemic stroke and magnetic resonance angiography (MRA) revealed focal narrowing in the M2 artery. An interesting finding is the restricted diffusion in the splenium of the corpus callosum, often observed in neonates with seizures. This may be accounted by the abundance of glutamate receptors in the corpus callosum of the developing brain, as well as homotopic axon projection from the right cerebral hemisphere to the left cerebral hemisphere.

Brain Development and Injury

To delineate the mechanisms of neonatal brain injury, it is important to consider the basic principles of brain development (also see Chapters 125 , 126 , and 132 ). The degree to which neuropsychiatric disease and neurodevelopmental disorders have a placental origin has been underestimated in the past. ^, Emerging evidence supports the concept of placental programming of conditions—including neuropsychiatric disease—that will be apparent in the postnatal and even adult life. Therefore, identifying the physiologic cellular trajectories of different types of neural cells in the developing brain will allow us to understand how the effect of multiple injurious insults might converge on a specific developmental time point vulnerable to conferring long-term disease impact.

The developmental processes of neuronal and glial cell lineage generation and maturation during gestation and neonatal life are complex and evolve during each gestational phase, peaking with rapid brain growth in the third trimester. During this critical period, the rapid cellular dynamics that contribute to the development of the brain also expose the vulnerability of maturational processes to injury. The brain weight, volume, and cortical surface increase rapidly during gestation, mainly between 23 and 35 weeks of gestation, with the formation of gyri and sulci. ^, The cerebellum lobules and folia are formed mostly during the last trimester of pregnancy. ^, ^, Indeed, third-trimester cerebellar growth is driven by sonic hedgehog (SHH) signaling, which can be directly inhibited by neonatal insults such as hypoxia and glucocorticoid exposure leading to cerebellar hypoplasia. Numerous fetal neuroimaging studies have highlighted the developmental changes and the complexity of the cerebral connectome during the last trimester of gestation. During this time and continuing into the first few years after birth, the brain undergoes several key events that lead to telencephalic organization. ^, These events include alignment, orientation, and lamination of cortical neurons; gyral development; dendritic arborization; synapse development; cell death; and elimination of specific neuronal processes and synapses that precede the formation of mature neural circuits. ^, ^,

Myelination is a critical process, initiated during fetal development and continuing postnatally into young adulthood. Myelin—the lipid-rich, tightly wrapped membranous extensions that ensheath axons—is essential for rapid communication within neural circuits and between different brain regions. The myelination process begins in the second trimester in caudal structures, such as the brain stem, and progresses rostrally towards the telencephalon in the third trimester. ^, Myelination requires the proliferation, migration, and subsequent differentiation of oligodendroglial cells, whose maturation culminates with myelin formation and wrapping around axons. ^, Oligodendrocyte progenitor cells (OPCs) and pre-oligodendrocytes (pre-OLs) proliferate (19 to 20 weeks), differentiate to mature OLs, which myelinate axonal processes (24 to 32 weeks) during different developmental waves. ^, Importantly, along with early-stage OLs, OPCs represent half of the total OL lineage cells at birth (40 weeks). Diseases that target myelin include multiple sclerosis (autoimmune) and Pelizaeus-Merzbacher disease (genetic), and both are associated with profound neurologic disability due to the lack of myelin function. Interestingly, work shows that myelin also includes channel proteins that help maintain axon integrity, suggesting that hypomyelination in preterm brain injury could be a secondary cause of axon and white matter volume decrease.

Etiology of Brain Injury in the Newborn

Brain injury in the fetal and neonatal period may be caused by numerous prenatal, perinatal, or postnatal factors ( Table 133.1 ) that manifest differently in neonates born at term or preterm. As most clinical signs are nonspecific, particularly during immediate postnatal presentation, differential diagnosis is often challenging. The following sections discuss:

1.
Mechanisms of brain injury , outlining the basic cellular and molecular mechanisms involved in neuronal injury following hypoxia-ischemia and inflammation. We will describe in detail the events following brain injury to include energy failure, excitotoxicity, free radical generation, growth factors, and other intracellular signaling leading to cell death.
2.
Clinical considerations in the neonate. We will review mechanistic perspectives related to specific clinical scenarios in the fetus and neonate starting in utero and extending into the neonatal period and infancy. We review the placental programming and the effect of prematurity on the developing brain. We will conclude this chapter by reviewing the mechanisms of injury in the neonatal brain caused by infection and inflammation, necrotizing enterocolitis (NEC), hyperbilirubinemia, and metabolic disorders, including hypoglycemia. We will also provide insights into future directions for neonatal brain research.

Table 133.1

Etiology of Neonatal Brain Injury.

Etiology of Neonatal Brain Injury	Clinical Pearls
1. Hypoxic-ischemic encephalopathy (HIE) and asphyxia	Total body hypothermia may improve neurodevelopment outcomes if initiated <6 h. Clinical trials on adjunctive therapies are underway
2. Infection (mainly meningitis and encephalitis)	Chorioamnionitis is a significant factor and is associated with poor neurodevelopment, often due to vertical transmission of neurotrophic viruses
3. Intraventricular hemorrhage (IVH)	Decreased incidence of IVH following various preventative measures in the NICU
4. Developmental brain malformations (neurulation, proliferation, migration defects)	Prenatal diagnostics, including fetal MRI, allow parents to engage in early decision making
5. Genetic syndromes	Advancements in preimplantation genetic testing (including free DNA testing) have increased the detection of genetic syndromes
6. Neonatal hypoglycemia	Insufficient evidence to support specific serum glucose levels associated with risk for brain injury, especially in the first 2 days of life
7. Birth trauma	Skull fracture, subdural, and subarachnoid or epidural hemorrhages associated with high morbidity
8. Placental insufficiency	Poor placental perfusion may be associated with IUGR and compromised neurodevelopment
9. Seizures	An acute sign of brain injury such as HIE, intracranial hemorrhage, or infection. Increasing evidence that seizures can worsen brain injury
10. Maternal stress	Preclinical and clinical evidence suggests an association with premature birth, epigenetic alterations in the placenta, IUGR, and poor neurodevelopment
11. Perinatal ischemic stroke (arterial or venous)	Unexplained etiology (>90%) with placental/cord infection a common suspect. Most commonly presents in the neonatal period (60%) or during early childhood (40%). If no underlying condition (e.g., cardiac disease, neural tube defect, or other), the recurrence risk <1%. Anticoagulation therapy remains controversial
12. Inborn errors of metabolism	Most present with pathognomonic patterns of brain injury using advanced imaging techniques, which may assist with early diagnosis and prompt initiation of treatments. In the majority of the acidemias the injury occurs after birth when maternal compensation is no longer possible. Neurodegenerative disorders can have onset in utero resulting in prenatal and progressive postnatal brain injury
13. Environmental toxicity (prenatal or postnatal)	Effects of lead toxicity well described—unclear impact of noise or benefit of private room configuration in the NICU on neurodevelopmental outcomes
14. Noxious substances	For example, alcohol (fetal alcohol syndrome), opiates, marijuana, cocaine (neonatal abstinence syndrome), SSRI (neonatal adaptive syndrome)

IUGR , Intrauterine growth restriction; MRI , magnetic resonance imaging; NICU , neonatal intensive care unit; SSRI , selective serotonin reuptake inhibitor.

Mechanisms of Brain Injury

Despite the multifactorial etiology of perinatal brain injury, with the exemption of the congenital and/or syndromic developmental brain malformations, many pathways converge to a common signaling cascade culminating in neuronal cell death. Therefore, in this chapter, we will be focusing on the cellular and molecular mechanisms of injury that follow specific pathologic events ( Fig. 133.2 ).

Fig. 133.2, Multifactorial causes and pleotropic effects characterize neonatal injury in the developing brain. The figure illustrates upstream causes, cascade events, and the impact in brain regions and at different developmental stages. The end result is either cell death of different neural cell populations, altered developmental trajectories, and/or brain circuits and function. ROS, Reactive oxygen species.

Energy Failure

The developing brain requires a constant supply of energy to initiate and complete developmental programs that include cell division, migration, differentiation, and biosynthesis of lipids, nucleotides, and proteins. While the mature brain primarily relies on glucose for its source of energy, the high metabolic demands of the rapidly developing brain utilize many substrates such as glucose, ketone bodies, lactate, fatty acids, and amino acids.

During early development, there are high circulating levels of lactate, ketone bodies, and glucose. The uptake and utilization of these substrates require transporters that are developmentally and regionally regulated. ^, Rodent studies have demonstrated that enzyme levels responsible for the oxidative metabolism of glucose exponentially increase during the first month after birth, while the proteins responsible for ketone metabolism are highly expressed in the first week and gradually decrease with age. ^,

Brain injury during the neonatal period is superimposed on the high metabolic demands of the developing brain. Acute injury, whether due to hypoxia, hypoxia-ischemia, or inflammation, can halt or interfere with normal developmental processes due to alterations in bioenergetic substrate availability, modifications in the expression of proteins/enzymes responsible for metabolism, and/or depletion of antioxidant capacity. The resulting energy failure can halt or delay normal maturation processes, contributing to neurodevelopmental delays.

Lactate and ketones enter the brain via specific monocarboxylate transporters (MCTs) and provide acetyl-CoA moieties that can directly enter the tricarboxylic acid (TCA) cycle. This process can provide more than half of the energy required during the first few weeks of life in rodents. Besides being a substrate for energy in the developing brain, ketones such as β-hydroxybutyrate may also decrease inflammatory cytokine release. Glucose enters the brain via specific glucose transporters that are also developmentally regulated. ^, As glycolysis provides a limited number of ATP molecules, the expression of enzymes necessary for oxidative metabolism—a significant contributor to ATP synthesis—exponentially increases during development. While glucose may not necessarily contribute to all of the cellular energy demands during the early stages of development, it does provide the carbon-backbone critical for nucleotide and NADPH synthesis via the pentose phosphate pathway (PPP). Studies have shown that the neonatal brain has a higher glucose flux into the PPP than the adult, which is consistent with the higher biosynthetic demands of early brain development. ^,

During the initial phase of a perinatal event, such as hypoxia, decreased oxygen, and glucose delivery, there is a switch in cellular anaerobic respiration. An initial decrease in phosphocreatine—the principal storage of high energy phosphate in the brain—first occurs, followed by decreased levels of ATP.

The accumulation of lactate and a decrease in pH initially lead to an increase in cerebral blood flow. However, a reduction in pH inhibits phosphofructokinase activity, resulting in decreased generation of ATP from glycolysis. Injured cells also experience an accumulation of excitotoxic amino acids—such as glutamate—and an increase in cytosolic Ca ²⁺ , combined with mitochondrial dysfunction and mitochondrial membrane permeabilization. The impact of the insult on mitochondria is clearly evident in changes in their dynamics—involving a balance between fission, mitophagy, fusion, and biogenesis—which normally ensure an adequate amount of ATP in the active cells. ^,

During periods of high energy ATP demands, including early development, fusion is necessary to create larger mitochondria and the mixing of mitochondrial contents. Following insult, such as oxygen-glucose deprivation, the balance of mitochondrial dynamics favors fission. ^, Damaged mitochondria undergo mitophagy (selective autophagy) through an autophagosomal-lysosomal pathway. The increased fission and mitophagy of mitochondria result in marked upregulation of mitochondrial DNA and proteins in the border zone regions of cortical infarcts at 24 hours after infarct.

After the initial phase of energy failure, there is a recovery of phosphocreatine levels with reduced lactate levels in the region. Using magnetic resonance (MR) spectroscopy, a few studies demonstrated that another decline in phosphocreatine occurs several hours later. ^,

Immunohistochemical studies also showed that this decline is preceded by a significant decrease in different neuronal cell populations. Therefore, evidence suggests that the observed decrease in phosphocreatine levels may be a consequence of events occurring during the primary energy failure phase, resulting in cell death.

The role of mitochondria in oxidative metabolism is not only to provide energy through ATP production, as the intermediate metabolites of the TCA cycle can produce either pro- or anti-inflammatory signals, and thus change the production of reactive oxygen species. The function of microglia, the nascent immune cells in the brain, may also be dictated by mitochondria and their dynamic state. In models of infection using lipopolysaccharide (LPS), an increase in mitochondrial-dependent oxidative phosphorylation in microglia was observed. However, prolonged LPS exposure decreased oxidative metabolism and increased glycolysis. ^,

Damaged mitochondria can also be pro-inflammatory because the released mitochondrial DNA acts as damage-associated molecular pattern (DAMP) molecules or binds to NLRP3. ^, These pathways promote autophagy, apoptosis, and disruption of glycolysis.

In conclusion, the maintenance of adequate energy levels is crucial for cell development and homeostasis. Without appropriate levels of energy substrates, the metabolic demands of a cell cannot be met, and a cascade of events occurs, which ultimately triggers the eventual demise of the cell. The developing brain is unique in that substrates other than glucose can be utilized, unlike the adult brain, where glucose is the primary source. Therefore, targeting metabolic pathways and mitochondria may lead to potential treatment regimens that restore energy balance during times of excessive metabolic demand imposed by neonatal brain injury.

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