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Dysmaturation of Premature Brain: Importance, Cellular Mechanisms, and Potential Interventions

  • Joseph J. Volpe
    Correspondence
    Communications should be addressed to: Volpe; Department of Pediatric Newborn Medicine; Brigham and Women's Hospital; 221 Longwood Avenue, Room 343C; Boston, MA 02115 USA.
    Affiliations
    Department of Neurology, Harvard Medical School, Boston, Massachusetts

    Department of Pediatric Newborn Medicine, Harvard Medical School, Boston, Massachusetts
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      Abstract

      Prematurity, especially preterm birth (less than 32 weeks' gestation), is common and associated with high rates of both survival and neurodevelopmental disability, especially apparent in cognitive spheres. The neuropathological substrate of this disability is now recognized to be related to a variety of dysmaturational disturbances of the brain. These disturbances follow initial brain injury, particularly cerebral white matter injury, and involve many of the extraordinary array of developmental events active in cerebral white and gray matter structures during the premature period. This review delineates these developmental events and the dysmaturational disturbances that occur in premature infants. The cellular mechanisms involved in the genesis of the dysmaturation are emphasized, with particular focus on the preoligodendrocyte. A central role for the diffusely distributed activated microglia and reactive astrocytes in the dysmaturation is now apparent. As these dysmaturational cellular mechanisms appear to occur over a relatively long time window, interventions to prevent or ameliorate the dysmaturation, that is, neurorestorative interventions, seem possible. Such interventions include pharmacologic agents, especially erythropoietin, and particular attention has also been paid to such nutritional factors as quality and source of milk, breastfeeding, polyunsaturated fatty acids, iron, and zinc. Recent studies also suggest a potent role for interventions directed at various experiential factors in the neonatal period and infancy, i.e., provision of optimal auditory and visual exposures, minimization of pain and stress, and a variety of other means of environmental behavioral enrichment, in enhancing brain development.

      Keywords

      Introduction

      Preterm birth (less than 37 weeks' gestation) is an enormous public health problem worldwide. According to the World Health Organization, approximately 15 million premature infants are born yearly and account for approximately one million deaths.
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      The United States ranks sixth among countries in terms of the number of preterm births. According to the Centers for Disease Control and Prevention, from 2014 to 2017 the preterm birth rate rose in the United States to approximately 10%. Of the approximately four million births in the United States, 1.4%, or about 56,000, are of very low birth weight (less than 1500 g).
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      Survival rates vary markedly as a function of gestational age but are at least 95% at 32 weeks', 90% at 28 weeks', and 60% to 65% at 24 weeks' gestation.
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      The substantial survival rates, unfortunately, are accompanied by relatively high incidences of neurological disability, for example, cerebral palsy in 5% to 10%, other motor disturbances in 25% to 40%, and cognitive, attentional, behavioral, and socialization disturbances in 25% to 50%.
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      The neuropathological substrate of this disability in preterm infants, especially those very preterm (less than 32 weeks' gestation) and extremely preterm (less than 28 weeks' gestation), consists of a combination of cerebral white matter injury (WMI) and especially, subsequent dysmaturational events in both white matter and neuroaxonal structures (see later). This combination of WMI and disturbances of gray matter structures has been termed the encephalopathy of prematurity.
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      Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances.
      In the initial review describing this encephalopathy, a particular emphasis was placed on the initial injury.
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      Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances.
      Subsequent work now suggests that although WMI is an important and likely initiating event, multiple subsequent dysmaturational events are most critical in determining the outcomes (see later). Moreover, because these dysmaturational events evolve over a very prolonged period (many months), a relatively long time window exists for interventions to prevent, counteract, or ameliorate the dysmaturation, i.e., neurorestorative interventions (see later).
      In the following discussion, I will review the multiple maturational events occurring in infant brain during the premature period; the dysmaturational events observed in premature infants, including the importance of the initiating cerebral WMI; the dysmaturational events that may occur without WMI; and the potential neuroprotective and neurorestorative interventions.

      Brain maturation during the premature period

      The brain dysmaturation that occurs in premature infants (see later) involves the multiple active developmental events occurring in human cerebrum during the period of 20 to 40 weeks' gestation and beyond. The rapidity and complexity of these cellular events underlie, to a considerable degree, their vulnerability to perturbations. The principal components involved include the oligodendroglial (OL) lineage, especially the preoligodendrocyte (pre-OL), cerebral white matter axons, subplate neurons, cerebral cortex, thalamus, and basal ganglia (Fig 1). In addition, microglia and astrocytes, especially in the white matter, are involved importantly in both normal development and dysmaturation of these principal components. The major developmental events during this period have been summarized in detail elsewhere.
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      • Volpe J.J.
      Organizational events.
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      • Volpe J.J.
      Myelination events.
      A brief review of the temporal aspects of these events is appropriate here (Table 1).
      Figure thumbnail gr1
      FIGURE 1Schematic of major components involved in brain maturation in the premature period. See text for details. GP, globus pallidus; Pre-OL, pre-oligodendrocyte; Put, putamen; SPN, subplate neuron; Thal, thalamus.
      TABLE 1Major Developmental Events During the Premature Period
      20-24 weeks
       Proliferation of OL progenitors
        Cerebral white matter axons (projection, commissural, and association) grow actively
       Subplate neuronal layer well established
       Thalamic afferent axons synapse abundantly on subplate neurons
      24-32 weeks
       OL progenitor differentiation leads to prominence of pre-OLs in cerebral white matter
       Cerebral white matter axons continue active growth
       Pre-OLs begin ensheathment of cerebral white matter axons
       Subplate reaches maximum size (several times thicker than cortical plate at 27-30 weeks)
       Thalamocortical afferent axons depart subplate neurons and enter cerebral cortex
       Cerebral cortical dendritic development and synaptogenesis become prominent
       Callosal (commissural) and association (corticocortical) axons enter subplate
       GABAergic neurons migrate into cerebral white matter
      32-40 weeks
       Pre-OLs remain the predominant cell of OL lineage in cerebral white matter until approximately 40 weeks when they and the more differentiated “immature” OL each account for approximately 50% of the OL lineage
       Subplate layer gradually decreases
       Callosal and corticocortical axons depart subplate and enter cerebral cortex
       GABAergic neurons migrate to cerebral cortex and populate upper cortical layers
       Cerebral cortical dendritic development and synaptogenesis become marked
      Abbreviation:
      OL = Oligodendroglial

      Pre-OL as a principal cellular target

      Pre-OLs are the principal cellular target in WMI of premature infants.
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      The developing oligodendrocyte: key cellular target in brain injury in the premature infant.
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      Encephalopathy of Prematurity: Pathophysiology.
      These cells are generated from OL progenitors and are the principal phase of the OL lineage during the premature period (Table 1, Fig 2). Pre-OLs account for 90% of the lineage during the peak period of WMI in premature infants. Even at term, pre-OLs account for 50% of the lineage in cerebral white matter, whereas approximately 50% of the lineage is the more differentiated “immature” OLs.
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      Late oligodendrocyte progenitors coincide with the developmental window of vulnerability for human perinatal white matter injury.
      Mature, myelin-producing OLs do not develop in human cerebral white matter to an appreciable degree until post-term. The pre-OL begins ensheathment of white matter axons at approximately 30 weeks' gestation (Fig 3).
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      Arrested oligodendrocyte lineage progression during human cerebral white matter development: Dissociation between the timing of progenitor differentiation and myelinogenesis.
      This process is critical for axonal differentiation
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      and, as a consequence, axonal function. The latter is the critical driving force for cerebral cortical development (see later), which evolves rapidly as an activity-dependent process during the third trimester of gestation.
      Figure thumbnail gr2
      FIGURE 2Major phases of the oligodendroglial lineage. The pre-OL (circled) is by far the predominant form during the premature period. OL, oligodendrocyte.
      Figure thumbnail gr3
      FIGURE 3Pre-OL ensheathment of axons at 30 weeks' gestational age. Pre-OL is immunostained green, and axon, red. OL, oligodendrocyte. (From Back SA, Luo NL, Borenstein NS, Volpe JJ, Kinney HC. Arrested oligodendrocyte lineage progression during human cerebral white matter development: Dissociation between the timing of progenitor differentiation and myelinogenesis. J Neuropathol Exp Neurol. 2002; 61:197-211, with permission).
      The pre-OL is a highly vulnerable cell, with particular susceptibility to such insults as hypoxia, ischemia, and inflammation, which lead to death via excitotoxic and free-radical-mediated mechanisms.
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      The developing oligodendrocyte: key cellular target in brain injury in the premature infant.
      The particular molecular characteristics that underlie this pre-OL vulnerability have been reviewed elsewhere.
      • Back S.A.
      • Volpe J.J.
      Encephalopathy of Prematurity: Pathophysiology.
      Suffice it to say here, many experimental studies of acute pre-OL death produced by hypoxia, ischemia, and inflammation have shown protective benefit for such agents as antiexcitotoxic, antiinflammatory, and antioxidant compounds (see later). Notably, however, as will be discussed later, in the premature infant with WMI, pre-OLs are replenished in the subacute period but fail to differentiate over the ensuing weeks or months to later phases of the OL lineage. As a result, hypomyelination is a hallmark of the disease.

      Axons

      Axonal development is remarkably active in the cerebrum during the premature period (and the early postnatal period) (Table 1).
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      Axonal development in the cerebral white matter of the human fetus and infant.
      Utilizing immunostaining with GAP-43, a protein expressed on growing axons, Haynes et al.
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      Axonal development in the cerebral white matter of the human fetus and infant.
      showed marked expression in cerebral white matter to at least 37 weeks' gestation. Growing white matter axons reach approximately the subplate region at 20 weeks, the deep layers of the cortical plate at 27 weeks, and the entire cortex by 37 weeks (Fig 4). Axonal growth occurs primarily within the cortex after 37 weeks and into the first year of life. Based on work by Kostovic and coworkers,
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      Correlation between the sequential ingrowth of afferents and transient patterns of cortical lamination in preterm infants.
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      Laminar organization of the human fetal cerebrum revealed by histochemical markers and magnetic resonance imaging.
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      the likely anatomic correlates of this progression in cerebral white matter during the premature period are growth of axons from thalamus to subplate neurons at 20 weeks and from subplate neurons to the cerebral cortex at 27 weeks (Table 1). Also, at 27 weeks, commissural and corticocortical cerebral white matter axons are actively growing, especially in the posterior periventricular regions, the so-called crossroads area. The increase in cerebral cortical expression of GAP-43 at 37 weeks may reflect a sum of continued cortical penetration from the subplate of thalamic ascending fibers and of commissural and corticocortical fibers (Fig 4). Thus it is apparent that the premature period is one of extraordinarily rapid axonal development, especially in cerebral white matter. Axons during this rapid growth period are exquisitely vulnerable to multiple insults (see later).
      Figure thumbnail gr4
      FIGURE 4GAP-43 expression in developing human parietal white matter and cortex. Cortex is indicated by an asterisk. Note at 20 postconceptional (PC) weeks (A) there is strong expression in cerebral white matter to a region below the cortical plate, likely subplate neurons. At 27 PC weeks (B) the expression begins to enter the cerebral cortex and continues in white matter. By 37 weeks (C), diffuse expression in cortex as well as continued expression in white matter are apparent. At 144 PC weeks (approximately age two years) (D), expression is prominent in cortex but not in white matter. (From Haynes RL, Borenstein NS, DeSilva TM, Folkerth RD, Liu LG, Volpe JJ, et al. Axonal development in the cerebral white matter of the human fetus and infant. J Comp Neurol. 2005; 484:156-67, with permission).

      Cerebral cortex-dendritic development, synaptogenesis

      The cerebral cortex undergoes dramatic changes during the premature period. These events include attainment of proper alignment, orientation and layering of cortical neurons (six layers apparent by 30 gestational weeks), arrival of late migrating GABAergic neurons (principally to upper cortical layers), elaboration of dendritic and axonal ramifications (neurite outgrowth), onset of synaptogenesis, and a marked increase in cortical surface area with gyral development (Table 1).
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      Organizational events.
      Neurite outgrowth, and particularly dendritic development, is most relevant in this context.
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      Ontogenesis of the pyramidal cell of the mammalian neocortex and developmental cytoarchitectonics: A unifying theory.
      Dendritic development is especially rapid in the third trimester (Fig 5) and is correlated with the development of cortical activity (Table 1).
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      Organizational events.
      Importantly, in this context the progress of dendritic development depends on the establishment of afferent input from cerebral white matter and then presumably synaptic activity.
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      Prenatal development of neurons in the human prefrontal cortex: I.A. qualitative Golgi study.
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      Nonsynaptic glycine receptor activation during early neocortical development.
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      Functional AMPA/kainate receptors in human embryonic and foetal central nervous system.
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      Dendrite development regulated by CREST, a calcium-regulated transcriptional activator.
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      Regulation of dendritic development by neuronal activity.
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      Activity-regulated transcription: bridging the gap between neural activity and behavior.
      Thus axonal input from subplate neurons and then from thalamic, commissural, and corticocortical fibers are the principal driving forces underlying cortical dendritic development.
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      Activity-regulated transcription: bridging the gap between neural activity and behavior.
      The importance of synaptogenesis in mediation of these effects of axonal input on cortical development has been emphasized in several studies (see
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      Timing in neural maturation: arrest, delay, precociousness, and temporal determination of malformations.
      for review). In a seminal study, Sarnat and coworkers studied synaptic development in human cerebral cortex from 6 to 41 weeks' gestational age with the immunomarker synaptophysin, which identified maturation of synaptic vesicles in axonal terminals. Thalamocortical axons exhibited intense staining in frontal cortex at approximately 26 weeks' gestation, and diffuse and uniformly strong staining was apparent throughout the cortex from 34 weeks onward.
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      Synaptophysin immunoreactivity in the human hippocampus and neocortex from 6 to 41 weeks of gestation.
      The findings integrate closely with measures of axonal development in the last trimester of gestation and with previous delineations of electrencephalographic maturation in premature infants. Functional synaptic activity via the axonal input to cortex is mediated principally through excitatory amino acid receptors, both the excitatory Ca++-permeable N-methyl-d-aspartate and GluR2-deficient α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, which exhibit exuberant expression in developing human cortex during this period.
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      Developmental regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. I. Rodent cerebral white matter and cortex.
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      Developmental regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. II. Human cerebral white matter and cortex.
      This role of functional activity has implications for the effects of a variety of environmental stimuli on cortical development in the premature infant, and for potential neurorestorative roles for such stimuli in the context of brain injury and dysmaturation (see later).
      Figure thumbnail gr5
      FIGURE 5Cerebral cortical development from 15 to 35 weeks' gestation, Golgi-Cox preparations. Magnification is the same for each sample. Note the remarkable apical and basilar dendritic development, especially after 24 weeks' gestation. (From Marin-Padilla M: Ontogenesis of the pyramidal cell of the mammalian neocortex and developmental cytoarchitectonics: A unifying theory, J Comp Neurol 321:233-240, 1992, with permission).

      Subplate neurons

      This important transient population of neurons is well-established in subcortical white matter by 20 weeks' gestation.
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      • Judas M.
      Correlation between the sequential ingrowth of afferents and transient patterns of cortical lamination in preterm infants.
      • Kostovic I.
      • Judas M.
      • Rados M.
      • et al.
      Laminar organization of the human fetal cerebrum revealed by histochemical markers and magnetic resonance imaging.
      • Kostovic I.
      • Jovanov-Milosevic N.
      The development of cerebral connections during the first 20-45 weeks' gestation.
      • Kostovic I.
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      Transient patterns of cortical lamination during prenatal life: do they have implications for treatment?.
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      During the important period of 24 to 32 weeks (the peak period for the occurrence of cerebral WMI) the subplate reaches maximum size (several times thicker than the cortical plate at 27 to 30 weeks) (Fig 1, Table 1). These neurons elaborate a dendritic arbor with spines, receive synaptic inputs from ascending afferents from thalamus and distant cortical sites,
      • Kinney H.C.
      • Volpe J.J.
      Organizational events.
      and extend axon collaterals to the overlying cerebral cortex and to other cortical and subcortical sites (thalamus, other cortical regions, corpus callosum). The crucial organizational functions of these neurons include provision of a transient synaptic site for ascending afferents from thalamus and other cortical sites, i.e., these “waiting” afferents cannot synapse yet in cortex because their neuronal targets have not yet differentiated. These afferents would undergo degeneration if they did not have the subplate neurons as transient targets. Moreover, the subplate neurons extend axons to cortex to promote cortical differentiation and to guide the afferent axons to cortex when sufficient cortical differentiation has occurred. Subplate axon collaterals also descend to pioneer or guide the initial axonal projections from cerebral cortex toward subcortical sites (e.g., thalamus, corpus callosum, other cortical sites). The subplate neuronal layer gradually decreases after 36 to 40 weeks' gestation.

      Late migrating GABAergic neurons

      Particularly characteristic of human cerebral cortical development is the relatively late generation of GABAergic neurons from the dorsal telencephalic subventricular zone and from the ventral ganglionic eminence (Fig 6).
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      Development of the human cerebral cortex: Boulder Committee revisited.
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      The origin of these late generated neurons is approximately 65% from the dorsal subventricular zone and 35% from the ventral ganglionic eminence. A substantial proportion of the ultimate population of GABAergic cortical neurons migrate through the cerebral white matter to the cortex in the third trimester. This migration peaks around term and then declines within the first six postnatal months.
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      Late development of the GABAergic system in the human cerebral cortex and white matter.
      Figure thumbnail gr6
      FIGURE 6GABAergic neuron development during the premature period. Proliferation of GABAergic neurons occurs in the dorsal subventricular zone (SVZ) and ventral ganglionic eminence; migration proceeds radially and tangentially to cortex and thalamus, as shown.

      Microglia and astrocytes

      Microglia and astrocytes are key players in the development of the white and gray matter structures just described. These glial elements also play a major causal role in the dysmaturational events that occur with cerebral WMI. Emphasis in this section is on the roles of microglia and astrocytes in normal development. Their role in dysmaturation is discussed later in the section on neuropathology.

      Microglia

      Microglia play important roles in such aspects of brain development such as axonal development, OL differentiation-myelination, vascularization, synaptogenesis, synaptic pruning, and neural circuit formation.
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      • et al.
      Single-cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes.
      The roles in OL development involve microglial proteins that stimulate pre-OL proliferation, enhance pre-OL survival and provide iron for OL differentiation, and secrete cytokines that enhance differentiation.
      • Hammond T.R.
      • Robinton D.
      • Stevens B.
      Microglia and the brain: complementary partners in development and disease.
      These cells are also the principal neuroimmune cells involved in neuroinflammatory responses. As part of the neuroinflammatory responses microglia can be destructive to cellular elements, such as pre-OLs, principally by generating free radicals, secreting injurious cytokines, and enhancing excitotoxicity (see later).
      • Hickman S.
      • Izzy S.
      • Sen P.
      • et al.
      Microglia in neurodegeneration.
      • Agresti C.
      • D'Urso D.
      • Levi G.
      Reversible inhibitory effects of interferon-γ- and tumour necrosis factor-α on oligodendroglial lineage cell proliferation and differentiation in vitro.
      • Andrews T.
      • Zhang P.
      • Bhat N.R.
      TNF-α potentiates IFNγ-induced cell death in oligodendrocyte progenitors.
      • Xie Z.
      • Wei M.
      • Morgan T.E.
      • et al.
      Peroxynitrite mediates neurotoxicity of amyloid β-peptide 1-42- and lipopolysaccharide-activated microglia.
      • Rivest S.
      Molecular insights on the cerebral innate immune system.
      • Lehnardt S.
      • Massillon L.
      • Follett P.
      • et al.
      Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway.
      • Buntinx M.
      • Moreels M.
      • Vandenabeele F.
      • et al.
      Cytokine-induced cell death in human oligodendroglial cell lines: I. Synergistic effects of IFN-gamma and TNF-alpha on apoptosis.
      • Li J.
      • Baud O.
      • Vartanian T.
      • et al.
      Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes.
      • Pang Y.
      • Cai Z.W.
      • Rhodes P.G.
      Effect of tumor necrosis factor-alpha on developing optic nerve oligodendrocytes in culture.
      • Larouche A.
      • Roy M.
      • Kadhim H.
      • et al.
      Neuronal injuries induced by perinatal hypoxic-ischemic insults are potentiated by prenatal exposure to lipopolysaccharide: animal model for perinatally acquired encephalopathy.
      Microglia have been characterized generally as pro-inflammatory (activated) (MI) or anti-inflammatory (M2). However, this bimodal characterization appears now to be too simplistic. Thus a recent landmark study in the developing mouse, utilizing molecular characterization methods, identified at least nine distinct microglial subpopulations with unique molecular signatures that changed over the course of development and exhibited marked spatial differences.
      • Hammond T.R.
      • Dufort C.
      • Dissing-Olesen L.
      • et al.
      Single-cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes.
      One distinct population was highly concentrated in axon tracts of the premyelinated brain. The molecular signatures of the microglial subpopulations in early development identified pathways associated with cell metabolism, growth, motility, and proliferation, among others. Studies in developing human brain will be of great interest.
      Microglia become prominent in the human forebrain at 16 to 22 weeks' gestation and migrate progressively through the white matter from 20 to 35 weeks, and then to the cerebral cortex.
      • Rezaie P.
      • Dean A.
      • Male D.
      • et al.
      Microglia in the cerebral wall of the human telencephalon at second trimester.
      • Rivest S.
      Molecular insights on the cerebral innate immune system.
      • Monier A.
      • Evrard P.
      • Gressens P.
      • et al.
      Distribution and differentiation of microglia in the human encephalon during the first two trimesters of gestation.
      • Billiards S.S.
      • Haynes R.L.
      • Folkerth R.D.
      • et al.
      Development of microglia in the cerebral white matter of the human fetus and infant.
      The critical point is that the cerebral white matter of the human premature infant is heavily populated with microglia during a period when various maturational events are occurring and when a variety of proinflammatory insults can lead to “activation” to destructive microglial phenotypes and WMI (see later). Moreover, because of the important role of microglial subpopulations in such important developmental events as OL development, axonal guidance, synaptogenesis, sculpting of neural networks, and cerebral connectivity, diversion of these normal cells to microglial phenotypes with primarily proinflammatory functions could contribute to disturbances in these maturational events observed in the premature brain.
      • Reemst K.
      • Noctor S.C.
      • Lucassen P.J.
      • et al.
      The indispensable roles of microglia and astrocytes during brain development.
      • Hickman S.
      • Izzy S.
      • Sen P.
      • et al.
      Microglia in neurodegeneration.

      Astrocytes

      The last half of human gestation also is a crucial time for astrocyte formation in the cerebrum.
      • Kinney H.C.
      • Volpe J.J.
      Organizational events.
      Fibrous astrocytes (generated from radial glial fibers) increasingly populate the cerebral white matter. During development astrocytes are important in axonal guidance, angiogenesis, formation of the blood-brain barrier, synaptogenesis, neuronal survival, and axonal and synaptic pruning.
      • Reemst K.
      • Noctor S.C.
      • Lucassen P.J.
      • et al.
      The indispensable roles of microglia and astrocytes during brain development.
      The molecular characteristics of astrocytes involved in facilitation of these events underlie such functions as expression of extracellular matrix (ECM) proteins and axonal guidance molecules, secretion of angiogenic factors, secretion of synaptogenesis molecules, clearance of extracellular glutamate, and secretion of various neurotrophic molecules. As will be discussed later, in the context of various brain insults (e.g., inflammation, hypoxia-ischemia), astrocytes can become “reactive” and exhibit a variety of metabolic changes that are deleterious to other white matter components, including pre-OLs.

      Dysmaturation in premature brain

      Overview

      The principal manifestations of dysmaturation in premature brain have been elucidated by advanced magnetic resonance imaging (MRI) techniques in living infants (Table 2). Briefly, the abnormalities have by volumetric MRI, diminished regional volumes, especially of cerebral cortex, white matter, thalamus, and basal ganglia; by diffusion-based imaging, in cerebral white matter, decreased fractional anisotropy (FA) with relatively greater involvement of radial diffusivity (consistent with impairment of pre-OL ensheathment of axons), and in cerebral cortex, blunting of the normal decline in FA (consistent with impaired dendritic development); by surface-based MRI measures, decreased cerebral cortical surface area and cortical folding or gyrification; and by functional MRI, impaired development of measures of connectivity, including especially thalamocortical connectivity. The abnormalities have been elucidated most commonly at term equivalent age, but generally persist, or may increase later in infancy, childhood, adolescence, or adulthood.
      • Zhang Y.
      • Inder T.E.
      • Neil J.J.
      • et al.
      Cortical structural abnormalities in very preterm children at 7years of age.
      • Smyser C.D.
      • Snyder A.Z.
      • Shimony J.S.
      • et al.
      Resting-state network complexity and magnitude are reduced in prematurely born infants.
      • Rajagopalan V.
      • Scott J.A.
      • Liu M.
      • et al.
      Complementary cortical gray and white matter developmental patterns in healthy, preterm neonates.
      • Batalle D.
      • Hughes E.J.
      • Zhang H.
      • et al.
      Early development of structural networks and the impact of prematurity on brain connectivity.
      • Neil J.J.
      • Smyser C.D.
      Recent advances in the use of MRI to assess early human cortical development.
      • Smyser C.D.
      • Wheelock M.D.
      • Limbrick Jr., D.D.
      • et al.
      Neonatal brain injury and aberrant connectivity.
      The most common accompaniment by MRI has been cerebral WMI (see later). The dysmaturational events, in general, appear to be secondary to WMI (see later discussion in Mechanics of dysmaturation with cerebral white matter injury). The constellation of WMI and the accompanying disturbances of neuronal or axonal structures is generally referred to as the encephalopathy of prematurity.
      • Volpe J.J.
      Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances.
      However, recent work suggests that some of the dysmaturational events documented in premature infants are not clearly related to WMI and perhaps are primary disturbances (see later). The emphasis in the following section is on the relation of cerebral WMI and dysmaturational events. Brief consideration of potentially primary dysmaturational events, perhaps independent of WMI, will then be presented.
      TABLE 2Dysmaturational Features in Premature Brain Elucidated by Advanced MRI
      Volumetric MRIDecreased regional volumes, especially cerebral cortex, white matter, thalamus, basal ganglia
      Diffusion imagingIn cerebral white matter, decreased FA, relatively increased radial diffusivity, variability altered axial diffusivity

      In cerebral cortex, blunting of the normal decline in FA
      Surface-based MRI measuresDecreased cerebral cortical surface area and cortical folding/gyrification
      Functional MRIImpaired development of measures of connectivity, including especially thalamocortical connectivity
      Abbreviations:
      FA = Fractional anisotropy
      MRI = Magnetic resonance imaging

      Cerebral white matter injury: A spectrum

      Neuropathology

      Cerebral WMI encompasses a spectrum of neuropathology that ranges from overt periventricular leukomalacia (PVL) to diffuse white matter gliosis (DWMG) (without focal necroses) (Fig 7A-C). The two fundamental characteristics of PVL are focal necroses with loss of all cellular elements in periventricular white matter and a more diffuse lesion in cerebral white matter, consisting initially of death of early differentiating pre-OLs, accompanied by vigorous and persistent astrogliosis and microgliosis (Fig 8).
      • Banker B.Q.
      • Larroche J.C.
      Periventricular leukomalacia of infancy. A form of neonatal anoxic encephalopathy.
      • Gilles F.H.
      Hypotensive brain stem necrosis. Selective symmetrical necrosis of tegmental neuronal aggregates following cardiac arrest.
      • Armstrong D.
      • Norman M.G.
      Periventricular leucomalacia in neonates. Complications and sequelae.
      • Shuman R.M.
      • Selednik L.J.
      Periventricular leukomalacia. A one-year autopsy study.
      • Okoshi Y.
      • Itoh M.
      • Takashima S.
      Characteristic neuropathology and plasticity in periventricular leukomalacia.
      • Kinney H.C.
      • Armstrong D.C.
      Perinatal neuropathology.
      • Pierson C.R.
      • Folkerth R.D.
      • Billards S.S.
      • et al.
      Gray matter injury associated with periventricular leukomalacia in the premature infant.
      • Buser J.R.
      • Maire J.
      • Riddle A.
      • et al.
      Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants.
      • Volpe J.J.
      Confusions in nomenclature: “periventricular leukomalacia” and “white matter injury” - identical, distinct, or overlapping?.
      The focal necroses are essentially infarcts. Temporally, in the more diffuse lesion the pre-OL disturbance consists acutely of cell death, followed subacutely and chronically initially by proliferation of pre-OLs but then critically, by a failure of maturation.
      • Haynes R.L.
      • Folkerth R.D.
      • Keefe R.
      • et al.
      Nitrosative and oxidative injury to premyelinating oligodendrocytes is accompanied by microglial activation in periventricular leukomalacia in the human premature infant.
      • Back S.A.
      • Luo N.L.
      • Mallinson R.A.
      • et al.
      Selective vulnerability of preterm white matter to oxidative damage defined by F(2)-isoprostanes.
      • Billiards S.S.
      • Haynes R.L.
      • Folkerth R.D.
      • et al.
      Myelin abnormalities without oligodendrocyte loss in periventricular leukomalacia.
      • Emery B.
      • Agalliu D.
      • Cahoy J.D.
      • et al.
      Myelin gene regulatory factor is a critical transcriptional regulator required for CNS myelination.
      • Fancy S.P.
      • Baranzini S.E.
      • Zhao C.
      • et al.
      Dysregulation of the Wnt pathway inhibits timely myelination and remyelination in the mammalian CNS.
      • Back S.A.
      White matter injury in the preterm infant: pathology and mechanisms.
      As noted earlier, this pre-OL dysmaturation underlies the subsequent hypomyelination, a unifying feature of PVL. The mildest form of WMI, i.e., DWMG without focal necroses, now is the most common form of WMI in premature infants and is also accompanied by the pre-OL dysmaturation.
      • Pierson C.R.
      • Folkerth R.D.
      • Billards S.S.
      • et al.
      Gray matter injury associated with periventricular leukomalacia in the premature infant.
      • Buser J.R.
      • Maire J.
      • Riddle A.
      • et al.
      Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants.
      • Volpe J.J.
      Confusions in nomenclature: “periventricular leukomalacia” and “white matter injury” - identical, distinct, or overlapping?.
      Figure thumbnail gr7
      FIGURE 7Spectrum of white matter injury (WMI) in premature infants. (A) to (C) illustrate the neuropathological spectrum of WMI. In severe WMI (A) the focal necrotic component consists of macroscopic areas of necrosis that result in cysts (i.e., “cystic” WMI). In moderate WMI (B) the focal necrotic component consists of small areas of necrosis that result in glial scars (i.e., “noncystic” WMI). In mild WMI (C) the focal component may be microscopic (less than 1 mm) or absent. In (A) through (C) the diffuse component of WMI consists of pre- oligodendrocyte (OL) injury or death (followed by pre-OL proliferation and maturation failure) and diffuse white matter gliosis (DWMG) involving activated microglia and reactive astrocytes. In panels (D) through (F) the magnetic resonance imaging correlates are shown. In (D), severe WMI, periventricular cysts are apparent; in (E), moderate WMI, punctate white matter lesions (PWMLs) but not cysts are seen; and in (F), mild WMI, only diffuse signal abnormality in white matter is apparent.
      Figure thumbnail gr8
      FIGURE 8Diffuse reactive astrogliosis (GFAP immunostain) and diffuse activation of microglia (CD-68 immunostain) in cerebral white matter in all three varieties of white matter injury (WMI) illustrated in . GFAP, glial fibrillary acidic protein.
      The relative distribution of the spectrum of cerebral WMI in the modern era has been delineated best by neuropathological studies. The two largest, most recent series demonstrate that compared with earlier studies the areas of focal necrosis are smaller and, indeed, most cases have few or none.
      • Pierson C.R.
      • Folkerth R.D.
      • Billards S.S.
      • et al.
      Gray matter injury associated with periventricular leukomalacia in the premature infant.
      • Buser J.R.
      • Maire J.
      • Riddle A.
      • et al.
      Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants.
      In the series by Pierson et al. (n = 41), true PVL, i.e., with focal necroses, occurred in 17 (42%). Importantly, nearly all of these lesions were less than 1 mm in size. In an additional 17 (42%), only DWMG without focal necroses was observed. (Only seven of the 41 brains were free of white matter abnormality.) Critically, Busser and coworkers observed in association with DWMG the sequelae of pre-OL death, i.e., the excess of pre-OLs and failure of pre-OL maturation.
      • Buser J.R.
      • Maire J.
      • Riddle A.
      • et al.
      Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants.
      Thus the full spectrum of cerebral WMI can be illustrated as shown in Fig 7A-C.

      Pathogenesis

      The pathogenesis of the focal necroses characteristic of PVL relates primarily to decreases in cerebral blood flow, related to a variety of perinatal or neonatal events, and the presence in the periventricular area of vascular border zones and end zones.
      • Volpe J.J.
      • Kinney H.C.
      • Jensen F.E.
      • et al.
      The developing oligodendrocyte: key cellular target in brain injury in the premature infant.
      • Back S.A.
      • Volpe J.J.
      Encephalopathy of Prematurity: Pathophysiology.
      The diffuse abnormality, DWMG, relates in considerable part to similar, albeit less severe clinical events (see later).
      In the diffuse lesion the pathogenesis of the acute pre-OL injury or death likely relates in part to the acute insults, noted above, as well as the accompanying disturbances that may predispose the pre-OL to injury (e.g., intrauterine growth retardation, systemic infection, impaired nutrition) (see later). The stimulus for the subsequent proliferative response of OL progenitors to produce abundant pre-OLs remains unclear. The pathogenesis of the subacute and chronic failure of maturation of these newly generated pre-OLs appears to relate to deleterious effects of the abundant activated microglia and reactive astrocytes characteristic of the diffuse lesion. These components likely are involved in other dysmaturational events relating to axonal and neuronal structures, as described next.

      Deleterious roles of microglia and reactive astrocytes

      The pro-inflammatory microglia may impair pre-OL maturation by release of reactive oxygen or nitrogen species or cytokines (e.g., tumor necrosis factor-α, interleukin [IL]-1β) that then act on pre-OLs.
      • Volpe J.J.
      • Kinney H.C.
      • Jensen F.E.
      • et al.
      The developing oligodendrocyte: key cellular target in brain injury in the premature infant.
      Recent studies of multiple sclerosis lesions have identified an inflammatory subpopulation of microglia that specifically targets myelin.
      • Hammond T.R.
      • Dufort C.
      • Dissing-Olesen L.
      • et al.
      Single-cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes.
      Whether such subpopulations are involved in pre-OL dysmaturation is unknown, but it is noteworthy that a large population of potentially activatable microglia are present in normal developing white matter during the premature period (see earlier). In addition, proinflammatory microglia have been shown recently to induce formation of neurotoxic reactive astrocytes.
      • Liddelow S.A.
      • Barres B.A.
      Reactive Astrocytes: Production, Function, and Therapeutic Potential.
      • Liddelow S.A.
      • Guttenplan K.A.
      • Clarke L.E.
      • et al.
      Neurotoxic reactive astrocytes are induced by activated microglia.
      As discussed next, such astrocytes are important in the pre-OL maturational failure. Finally, the shift in microglial phenotype from an antiinflammatory to a proinflammatory activated phenotype diverts the critical roles of “normal” microglia in OL development described earlier.
      The abundant “reactive” astrocytes (A1) in DWMG also likely play critical roles in the failure of pre-OL maturation.
      • Back S.A.
      White matter injury in the preterm infant: pathology and mechanisms.
      • Liddelow S.A.
      • Barres B.A.
      Reactive Astrocytes: Production, Function, and Therapeutic Potential.
      • Liddelow S.A.
      • Guttenplan K.A.
      • Clarke L.E.
      • et al.
      Neurotoxic reactive astrocytes are induced by activated microglia.
      • Cekanaviciute E.
      • Buckwalter M.S.
      Astrocytes: integrative regulators of neuroinflammation in stroke and other neurological diseases.
      The best established mechanism in the context of WMI is based on seminal work by Back and coworkers.
      • Buser J.R.
      • Maire J.
      • Riddle A.
      • et al.
      Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants.
      • Back S.A.
      White matter injury in the preterm infant: pathology and mechanisms.
      • Cargill R.
      • Kohama S.G.
      • Struve J.
      • et al.
      Astrocytes in aged nonhuman primate brain gray matter synthesize excess hyaluronan.
      The likely sequence involves the generation by reactive astrocytes of high-molecular-weight forms of hyaluronic acid. Astrocyte-associated ECM is also involved in this generation. ECM is also a key source of hyaluronidases, which convert the high-molecular-weight forms of hyaluronic acid to lower-molecular-weight forms. The latter lead to failure of pre-OL maturation, probably by activating Toll-like receptor-2 receptors on pre-OLs.
      • Sloane J.A.
      • Batt C.
      • Ma Y.
      • et al.
      Hyaluronan blocks oligodendrocyte progenitor maturation and remyelination through TLR2.
      The particular role of hyaluronan is supported by the observation that pharmacologic inhibition of hyaluronidases promotes pre-OL maturation and myelination (see later). Other products of reactive astrocytes may also be involved in the pre-OL dysmaturation. Thus, in human WMI reactive astrocytes express large amounts of interferon-γ, and pre-OLs express the interferon-γ receptor,
      • Folkerth R.D.
      • Keefe R.J.
      • Haynes R.L.
      • et al.
      Interferon-gamma expression in periventricular leukomalacia in the human brain.
      activation of which inhibits pre-OL differentiation.
      • Volpe J.J.
      • Kinney H.C.
      • Jensen F.E.
      • et al.
      The developing oligodendrocyte: key cellular target in brain injury in the premature infant.
      Other products of astrocytes may contribute to the inhibition of pre-OL differentiation, for example, bone morphogenetic proteins and Notch ligand Jagged 1, but data on human preterm WMI are not yet available.
      • Back S.A.
      White matter injury in the preterm infant: pathology and mechanisms.
      Finally, as noted for activated microglia, the shift in astrocyte phenotype from normal fibrous astrocytes to the toxic reactive phenotype also diverts the critical roles of astrocytes in the development of OLs (see earlier).
      In view of the apparent critical roles of activated microglia and reactive astrocytes in disturbing pre-OL development (and likely also, aspects of axonal development), the question of the duration of DWMG in cerebral WMI of the premature infant becomes critical. Thus available evidence by MRI in vivo suggests that dysmaturation continues for many months and likely longer. Not unexpectedly, neuropathological data in human infants concerning duration of DWMG are somewhat scanty. However, available information suggests that DWMG is present for at least many months after the premature period and likely longer.
      • Okoshi Y.
      • Itoh M.
      • Takashima S.
      Characteristic neuropathology and plasticity in periventricular leukomalacia.
      • Pierson C.R.
      • Folkerth R.D.
      • Billards S.S.
      • et al.
      Gray matter injury associated with periventricular leukomalacia in the premature infant.
      • Haynes R.L.
      • Folkerth R.D.
      • Keefe R.
      • et al.
      Nitrosative and oxidative injury to premyelinating oligodendrocytes is accompanied by microglial activation in periventricular leukomalacia in the human premature infant.
      • Deguchi K.
      • Oguchi K.
      • Takashima S.
      Characteristic neuropathology of leukomalacia in extremely low birth weight infants.
      There is precedent for microglia to be chronically activated in human neuropathology, for example, after traumatic brain injury.
      • Donat C.K.
      • Scott G.
      • Gentleman S.M.
      • et al.
      Microglial activation in traumatic brain injury.
      In the latter setting, these cells are considered important in subsequent degeneration of axons and neurons years later and to play a role in the enhanced incidence of degenerative disorders, such as Alzheimer and Parkinson diseases.

      Identification in vivo

      Neuroradiological identification of the cerebral WMI spectrum in vivo is made best by MRI but is not entirely satisfactory. Thus the most severe end of the WMI spectrum, i.e., severe WMI, with large areas of necrosis and apparent cystic change, are readily identified as such (Fig 7D). However, such lesions are observed on MRI (and by neuropathology) in less than 5% of infants in modern neonatal intensive care facilities.
      • Kwon S.H.
      • Vasung L.
      • Ment L.R.
      • et al.
      The role of neuroimaging in predicting neurodevelopmental outcomes of preterm neonates.
      • Neil J.J.
      • Volpe J.J.
      Encephalopathy of prematurity: Clinical-neurological features, diagnosis, imaging, prognosis, therapy.
      More common are small areas of necrosis (more than 1 mm) in periventricular and central cerebral white matter, seen at term equivalent age as (noncystic) punctate white matter lesions (PWMLs) in 15% to 25%, i.e., moderate WMI (Fig 7E).
      • Chau V.
      • Synnes A.
      • Grunau R.E.
      • et al.
      Abnormal brain maturation in preterm neonates associated with adverse neurodevelopmental outcomes.
      • Kersbergen K.J.
      • Benders M.J.
      • Groenendaal F.
      • et al.
      Different patterns of punctate white matter lesions in serially scanned preterm infants.
      • Guo T.
      • Duerden E.G.
      • Adams E.
      • et al.
      Quantitative assessment of white matter injury in preterm neonates: Association with outcomes.
      • Tusor N.
      • Benders M.J.
      • Counsell S.J.
      • et al.
      Punctate white matter lesions associated with altered brain development and adverse motor outcome in preterm infants.
      Notably, this incidence of noncystic PVL (PWMLs) is appreciably higher if scans are performed early in the neonatal period—presumably the gliotic scars contract sufficiently to become invisible to MRI by term equivalent age. The least severe end of the WMI spectrum, i.e., mild WMI, is likely a heterogeneous group. Thus the large majority of focal necroses observed postmortem are less than 1 mm in size
      • Pierson C.R.
      • Folkerth R.D.
      • Billards S.S.
      • et al.
      Gray matter injury associated with periventricular leukomalacia in the premature infant.
      • Buser J.R.
      • Maire J.
      • Riddle A.
      • et al.
      Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants.
      and likely below the resolution of most conventional MRI scanners. In addition, the MRI correlate of the very common DWMG, without focal necroses, also is unknown. Importantly, as with the diffuse gliotic component of overt PVL, DWMG alone appears to lead to pre-OL death and subsequent dysmaturation,
      • Buser J.R.
      • Maire J.
      • Riddle A.
      • et al.
      Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants.
      and thus may be very important clinically. The frequent isolated finding of diffuse signal abnormality in cerebral white matter (Fig 7F) may be the MRI correlate of mild WMI, although both the reproducibility of this imaging finding and the relation to outcome remain unclear.
      • Volpe J.J.
      Confusions in nomenclature: “periventricular leukomalacia” and “white matter injury” - identical, distinct, or overlapping?.
      The few excellent studies that identify WMI without detectable focal necroses by the presence of diminished FA on diffusion-based MRI (see later) may be the best in vivo correlate of the admixture of the WMI spectrum that includes the two large groups of (1) focal necroses too small for identification (with DWMG) and (2) DWMG without any focal necroses, the two forms that we refer to as mild WMI.

      Clinical importance

      The clinical importance of the cerebral WMI spectrum relates to the motor and cognitive deficits associated with the lesion and the subsequent dysmaturation. The clinical phenomena associated with moderate and severe WMI have been described in detail elsewhere.
      • Neil J.J.
      • Volpe J.J.
      Encephalopathy of prematurity: Clinical-neurological features, diagnosis, imaging, prognosis, therapy.
      Identification of the neurodevelopmental sequelae of mild WMI is hindered by the difficulty in identifying the lesion by conventional MRI, as used in most large-scale studies. Large-scale MRI studies of premature infants show, as expected, worsening clinical outcomes as a function of severity of WMI. However, it is noteworthy that infants with either no or “mild” abnormality in cerebral white matter by conventional MRI still exhibit neurological disability subsequently. Although cognitive scales utilized among studies vary, cognitive scores for infants (less than 28 to 30 weeks' gestation) with no or “mild” WMI are approximately 85 to 93.
      • Guo T.
      • Duerden E.G.
      • Adams E.
      • et al.
      Quantitative assessment of white matter injury in preterm neonates: Association with outcomes.
      • Woodward L.J.
      • Anderson P.J.
      • Austin N.C.
      • et al.
      Neonatal MRI to predict neurodevelopmental outcomes in preterm infants.
      • Hintz S.R.
      • Barnes P.D.
      • Bulas D.
      • et al.
      Neuroimaging and neurodevelopmental outcome in extremely preterm infants.
      • Linsell L.
      • Johnson S.
      • Wolke D.
      • et al.
      Cognitive trajectories from infancy to early adulthood following birth before 26 weeks of gestation: a prospective, population-based cohort study.
      In a particularly well-characterized study of 480 extremely preterm infants (less than 28 weeks' gestation), 20% of infants with no apparent WMI by conventional MRI had cognitive scores less than 85.
      • Hintz S.R.
      • Barnes P.D.
      • Bulas D.
      • et al.
      Neuroimaging and neurodevelopmental outcome in extremely preterm infants.
      The possibility that neuroaxonal dysmaturation with mild WMI (see mechanisms of dysmaturation with cerebral white matter injury) is important in determination of these outcomes is suggested by follow-up studies that included assessment of gray matter abnormalities (as well as WMI).
      • Anderson P.J.
      • Treyvaud K.
      • Neil J.J.
      • et al.
      Associations of newborn brain magnetic resonance imaging with long-term neurodevelopmental impairments in very preterm children.
      As will be discussed later, studies that assess WMI by highly sensitive diffusion MRI measures show a clear association between mild WMI, dysmaturation, and subsequent cognitive disturbances.

      Mechanisms of dysmaturation with cerebral white matter injury

      The mechanisms of the dysmaturational features identified by MRI in premature brains (Table 2), especially in the context of WMI, are likely multiple. The prevailing theme is a sequence whereby the initial insult (hypoxia, ischemia, inflammation, infection, etc.) leads to primary cellular injury or death, which in turn results in the subsequent replenishment of pre-OLs but secondary dysmaturation. The cellular elements injured likely depend on the severity of the WMI. Thus in moderate to severe WMI (Fig 7A,B), all the rapidly developing cellular elements, as outlined next, appear to be injured, whereas in mild WMI (Fig 7C), the pre-OL may be the principal or only cellular element undergoing primary injury.

      Dysmaturation with moderate-severe white matter injury

      Pre-OL injury

      Primary injury or death of the pre-OL, which is exquisitely vulnerable to hypoxic-ischemic, inflammatory, or related insults, is a consistent early feature of all forms of WMI.
      • Back S.A.
      Brain injury in the preterm infant: New horizons for pathogenesis and prevention.
      • Back S.A.
      White matter injury in the preterm infant: pathology and mechanisms.
      • Kinney H.C.
      • Volpe J.J.
      Encephalopathy of Prematurity: Neuropathology.
      Cell death, irreversible process loss, or both have been documented acutely.
      • Back S.A.
      Brain injury in the preterm infant: New horizons for pathogenesis and prevention.
      • Haynes R.L.
      • Folkerth R.D.
      • Keefe R.
      • et al.
      Nitrosative and oxidative injury to premyelinating oligodendrocytes is accompanied by microglial activation in periventricular leukomalacia in the human premature infant.
      • Billiards S.S.
      • Haynes R.L.
      • Folkerth R.D.
      • et al.
      Myelin abnormalities without oligodendrocyte loss in periventricular leukomalacia.
      • Back S.A.
      White matter injury in the preterm infant: pathology and mechanisms.
      • Kinney H.C.
      • Volpe J.J.
      Encephalopathy of Prematurity: Neuropathology.
      Subsequently, over the ensuing weeks replenishment of the pre-OL pool occurs but subsequent maturation to mature, myelin-producing OLs fails. The important role of reactive astrocytes and activated microglia in this dysmaturation was described earlier. The result of this pre-OL dysmaturation is hypomyelination (Fig 9A). Also, however, pre-OL dysmaturation likely leads to failure of pre-OL ensheathment of axons, and as a consequence, impaired development, i.e., dysmaturation, of axons. The important trophic role of pre-OLs for axonal development, survival, and function was noted earlier. Indeed, this process is likely crucial for the exuberant axonal growth in cerebral white matter illustrated earlier (Fig 4) and the activity-dependent development of cerebral cortex (Fig 5). The consequences of the axonal disturbance would be diminished volumes of cerebral cortex and thalamus or basal ganglia, secondary to retrograde and anterograde (trans-synaptic) effects, i.e., involving projection fibers to and from the cortex, thalamus, and basal ganglia, i.e., thalamocortical, corticospinal, corticostriatal, and commissural and association fibers to and from the cortex, i.e., corticocortical (Fig 9A).
      Figure thumbnail gr9
      FIGURE 9Mechanisms of dysmaturation following injury to pre- oligodendrocytes (OLs) (A), axons (B), thalamus (C), subplate neurons, (D) or migrating GABAergic neurons (E). For sequences (A–D), the initial injury leads to multiple dysmaturational events involving pre-OLs and axons. The principal outcomes are the disturbances of myelination and cortical and thalamic development, as shown in vivo by magnetic resonance imaging. See text for details.

      Axonal injury

      Primary injury to the rapidly developing, vulnerable, premyelinating axons in cerebral white matter could be a primary event with WMI (Fig 9B). Although axonal injury is shown readily in the areas of focal necrosis, a more widespread degeneration of axons detected by the apoptotic marker, fractin, also has been identified.
      • Haynes R.L.
      • Billiards S.S.
      • Borenstein N.S.
      • et al.
      Diffuse axonal injury in periventricular leukomalacia as determined by apoptotic marker fractin.
      This finding is consistent with related experimental observations concerning the vulnerability of developing axons.
      • Tekkok S.B.
      • Goldberg M.P.
      AMPA/kainate receptor activation mediates hypoxic oligodendrocyte death and axonal injury in cerebral white matter.
      • Wakita H.
      • Tomimoto H.
      • Akiguchi I.
      • et al.
      Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat.
      • Sizonenko S.V.
      • Sirimanne E.
      • Mayall Y.
      • et al.
      Selective cortical alteration after hypoxic-ischemic injury in the very immature rat brain.
      • McCarran W.J.
      • Goldberg M.P.
      White matter axon vulnerability to AMPA/kainate receptor-mediated ischemic injury is developmentally regulated.
      • Alix J.J.
      • Zammit C.
      • Riddle A.
      • et al.
      Central axons preparing to myelinate are highly sensitivity to ischemic injury.
      The dysmaturational events subsequent to axonal injury (Fig 9B) by anterograde and retrograde effects would result in the impairments of cortical and thalamic development and related abnormalities detected by MRI (see Table 2). An impairment of pre-OL maturation would result from the loss of trophic axonal signals, with the ultimate consequence, hypomyelination. A contributory role for deleterious effects of activated microglia and reactive astrocytes (see earlier) also seems likely. Moreover, because of the role of both these glial types in normal axonal development, diversion to activated or reactive phenotypes may further impair axonal development.

      Thalamic injury

      Primary injury to thalamus is suggested by a neuropathological study of human infants with moderate to severe WMI and thalamic abnormalities (neuronal loss, gliosis, axonal degeneration) detected in approximately 60%.
      • Pierson C.R.
      • Folkerth R.D.
      • Billards S.S.
      • et al.
      Gray matter injury associated with periventricular leukomalacia in the premature infant.
      • Ligam P.
      • Haynes R.L.
      • Folkerth R.D.
      • et al.
      Thalamic damage in periventricular leukomalacia: Novel pathologic observations relevant to cognitive deficits in survivors of prematurity.
      A particular vulnerability of thalamus has also been shown in an experimental model.
      • Northington F.J.
      • Ferriero D.M.
      • Flock D.L.
      • et al.
      Delayed neurodegeneration in neonatal rat thalamus after hypoxia-ischemia is apoptosis.
      • Northington F.J.
      • Ferriero D.M.
      • Martin L.J.
      Neurodegeneration in the thalamus following neonatal hypoxia-ischemia is programmed cell death.
      Primary injury to thalamus could lead to degeneration of axons originating and terminating in the thalamus and, as a consequence, to pre-OL dysmaturation and hypomyelination (Fig 9C).

      Subplate neuronal injury

      Primary injury to subplate neurons would be expected to have major secondary dysmaturational effects on thalamus by retrograde degenerative effects on ascending thalamic axons (“waiting afferents”), as well as on cerebral cortex by anterograde effects via loss of subplate neuronal axons to cortex and on descending cortical axonal projections by loss of guidance from subplate axonal collaterals (Fig 9D). Considerable experimental data support these contentions.
      • Kostovic I.
      • Judas M.
      Correlation between the sequential ingrowth of afferents and transient patterns of cortical lamination in preterm infants.
      • McConnell S.K.
      • Ghosh A.
      • Shatz C.J.
      Subplate neurons pioneer the first axon pathway from the cerebral cortex.
      • Ghosh A.
      • Antonini A.
      • McConnell S.K.
      • et al.
      Requirement for subplate neurons in the formation of thalamocortical connections.
      • Ghosh A.
      • Shatz C.J.
      Involvement of subplate neurons in the formation of ocular dominance columns.
      • Ghosh A.
      • Shatz C.J.
      A role for subplate neurons in the patterning of connections from thalamus to neocortex.
      • Volpe J.J.
      Subplate neurons - missing link in brain injury of the premature infant?.
      • Kanold P.O.
      • Kara P.
      • Reid R.C.
      • et al.
      Role of subplate neurons in functional maturation of visual cortical columns.
      • Kanold P.O.
      Transient microcircuits formed by subplate neurons and their role in functional development of thalamocortical connections.
      • Bystron I.
      • Molnar Z.
      • Otellin V.
      • et al.
      Tangential networks of precocious neurons and early axonal outgrowth in the embryonic human forebrain.
      With axonal degeneration, subsequent disturbances in pre-OL development would be expected (Fig 9D). Although data are not entirely consistent, experimental studies suggest that subplate neurons are particularly vulnerable to hypoxia-ischemia.
      • McQuillen P.S.
      • Sheldon R.A.
      • Shatz C.J.
      • et al.
      Selective vulnerability of subplate neurons after early neonatal hypoxia-ischemia.
      Two reports suggest a loss of subplate neurons in premature infants with moderate to severe WMI.
      • Robinson S.
      • Li Q.
      • Dechant A.
      • et al.
      Neonatal loss of gamma-aminobutyric acid pathway expression after human perinatal brain injury.
      • Kinney H.C.
      • Haynes R.L.
      • Xu G.
      • et al.
      Neuron deficit in the white matter and subplate in periventricular leukomalacia.

      Late migrating GABAergic neurons

      Primary injury to late migrating GABAergic neurons seems possible because the migratory path of these late generated cells is from the dorsal subventricular zone through cerebral white matter to the cerebral cortex (Fig 6). Two neuropathological studies of moderate to severe WMI show a deficit in central white matter neurons consistent with late migrating GABAergic neurons.
      • Robinson S.
      • Li Q.
      • Dechant A.
      • et al.
      Neonatal loss of gamma-aminobutyric acid pathway expression after human perinatal brain injury.
      • Kinney H.C.
      • Haynes R.L.
      • Xu G.
      • et al.
      Neuron deficit in the white matter and subplate in periventricular leukomalacia.
      The result of a disturbance in these neurons would be a deficit in cerebral cortical neurons, especially the upper cortical layers (Fig 9E). The MRI result would be diminished cerebral cortical volume, surface area, gyrification, and connectivity, as noted in advanced MRI studies (Table 2).

      Conclusions

      Thus, in moderate to severe WMI, i.e., identified by neonatal MRI by PWMLs (relatively common) or by cystic lesions (rare), several potential sequences of primary injury leading to dysmaturation and developmental impairments detected by advanced MRI techniques seem likely. Although pre-OL death and subsequent replenishment of pre-OLs, which then fail to mature, appear most consistent (Fig 9A), the other sequences depicted in Fig 9 may also occur to varying degrees, dependent in part on such factors as the gestational age of the infant; the nature, severity, and timing of the initiating insult(s); and the presence of other potentiating factors, for example, intrauterine adversity, postnatal infection, undernutrition, etc.

      Dysmaturation with mild white matter injury

      Mild WMI, as discussed earlier, is characterized by focal necrotic lesions less than approximately 1 mm in size, and thus undetectable by conventional MRI, or DWMG without focal necroses (Fig 7C). The dysmaturational features apparent subsequently in vivo by advanced MRI are similar in many respects to those described earlier for moderate and severe WMI (Table 2) but are less pronounced.
      • Neil J.J.
      • Volpe J.J.
      Encephalopathy of prematurity: Clinical-neurological features, diagnosis, imaging, prognosis, therapy.
      • Boardman J.P.
      • Counsell S.J.
      • Rueckert D.
      • et al.
      Abnormal deep grey matter development following preterm birth detected using deformation based morphometry.
      • Anjari M.
      • Srinivasan L.
      • Allsop J.M.
      • et al.
      Diffusion tensor imaging with tract-based spatial statistics reveals local white matter abnormalities in preterm infants.
      • Dubois J.
      • Benders M.
      • Cachia A.
      • et al.
      Mapping the early cortical folding process in the preterm newborn brain.
      • Boardman J.P.
      • Craven C.
      • Valappil S.
      • et al.
      A common neonatal image phenotype predicts adverse neurodevelopmental outcome in children born preterm.
      • Ball G.
      • Boardman J.P.
      • Rueckert D.
      • et al.
      The effect of preterm birth on thalamic and cortical development.
      • Ball G.
      • Boardman J.P.
      • Aljabar P.
      • et al.
      The influence of preterm birth on the developing thalamocortical connectome.
      • Ball G.
      • Aljabar P.
      • Nongena P.
      • et al.
      Multimodal image analysis of clinical influences on preterm brain development.
      • Barnett M.L.
      • Tusor N.
      • Ball G.
      • et al.
      Exploring the multiple-hit hypothesis of preterm white matter damage using diffusion MRI.
      Thus a series of careful studies of premature infants without major WMI and utilizing diffusion-based MRI determinations of FA and related measures in cerebral white matter as a means to detect mild WMI, not readily apparent on conventional MRI, show at term equivalent age disturbances in volumetric development of cerebral cortex, cerebral white matter, thalamus, basal ganglia, cortical folding, cortical and white matter microstructure, and thalamocortical connectivity.
      • Boardman J.P.
      • Counsell S.J.
      • Rueckert D.
      • et al.
      Abnormal deep grey matter development following preterm birth detected using deformation based morphometry.
      • Anjari M.
      • Srinivasan L.
      • Allsop J.M.
      • et al.
      Diffusion tensor imaging with tract-based spatial statistics reveals local white matter abnormalities in preterm infants.
      • Dubois J.
      • Benders M.
      • Cachia A.
      • et al.
      Mapping the early cortical folding process in the preterm newborn brain.
      • Boardman J.P.
      • Craven C.
      • Valappil S.
      • et al.
      A common neonatal image phenotype predicts adverse neurodevelopmental outcome in children born preterm.
      • Ball G.
      • Boardman J.P.
      • Rueckert D.
      • et al.
      The effect of preterm birth on thalamic and cortical development.
      • Ball G.
      • Boardman J.P.
      • Aljabar P.
      • et al.
      The influence of preterm birth on the developing thalamocortical connectome.
      • Barnett M.L.
      • Tusor N.
      • Ball G.
      • et al.
      Exploring the multiple-hit hypothesis of preterm white matter damage using diffusion MRI.
      • Ajayi-Obe M.
      • Saeed N.
      • Cowan F.M.
      • et al.
      Reduced development of cerebral cortex in extremely preterm infants.
      • Ball G.
      • Pazderova L.
      • Chew A.
      • et al.
      Thalamocortical connectivity predicts cognition in children born preterm.
      In a particularly large, recent series (n = 491), Barnett et al. identified lower FA in cerebral white matter with particularly high radial (versus axial) diffusion (RD).
      • Barnett M.L.
      • Tusor N.
      • Ball G.
      • et al.
      Exploring the multiple-hit hypothesis of preterm white matter damage using diffusion MRI.
      The high RD is consistent with an impairment of pre-OL ensheathment.
      • Neil J.J.
      • Volpe J.J.
      Encephalopathy of prematurity: Clinical-neurological features, diagnosis, imaging, prognosis, therapy.
      • Wimberger D.M.
      • Roberts T.P.
      • Barkovich A.J.
      • et al.
      Identification of “premyelination” by diffusion-weighted MRI.
      The findings suggest that impaired pre-OL maturation is the critical finding in mild cerebral WMI. The lower FA values were independently associated with increased number of days on ventilation, perhaps consistent with chronic hypoxia or related insults and with fetal growth restriction. The latter has been shown to be associated with a degree of hypoxia and in experimental studies to lead to delayed OL maturation
      • Barnett M.L.
      • Tusor N.
      • Ball G.
      • et al.
      Exploring the multiple-hit hypothesis of preterm white matter damage using diffusion MRI.
      • Tolcos M.
      • Bateman E.
      • O'Dowd R.
      • et al.
      Intrauterine growth restriction affects the maturation of myelin.
      —recall that the pre-OL is exquisitely vulnerable to hypoxic and related insults (see earlier). The white matter findings also related to prolonged parenteral nutrition and suggest that impaired nutrition may lead to impaired pre-OL development (see later). Importantly, the abnormal FA values in the large study of Barnett et al. were associated with impaired neurodevelopmental performance at age 20 months.
      • Barnett M.L.
      • Tusor N.
      • Ball G.
      • et al.
      Exploring the multiple-hit hypothesis of preterm white matter damage using diffusion MRI.
      As noted earlier, two major neuropathological series indicate that mild WMI, as defined here, is at present the dominant form of cerebral WMI.
      • Pierson C.R.
      • Folkerth R.D.
      • Billards S.S.
      • et al.
      Gray matter injury associated with periventricular leukomalacia in the premature infant.
      • Buser J.R.
      • Maire J.
      • Riddle A.
      • et al.
      Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants.
      As noted earlier, detection of this milder but prevalent form of WMI cannot be made consistently by conventional MRI. The recent work just described with diffusion-based MRI indicates promise for detection in vivo.
      Although the mechanisms for dysmaturation with mild WMI may overlap with those just described for moderate to severe WMI, major differences are likely. Thus, with mild WMI clear evidence for primary injury to components other than the pre-OL is lacking. It is most likely that with mild WMI the deleterious effects of the abundant activated microglia and reactive astrocytes are the dominant mediators of dysmaturation, especially to the pre-OL, and perhaps also to axons.

      Pre-OL injury

      Primary injury or death of the pre-OL with subsequent replenishment of pre-OLs but failure of maturation, as described for moderate to severe WMI (see earlier), may be the major mechanism for the widespread dysmaturation just described. The important role of activated microglia and reactive astrocytes was discussed earlier concerning moderate to severe WMI. The scenario to widespread dysmaturation, thus, would be similar to that described for moderate to severe WMI (Fig 9A).

      Axonal injury

      Although evidence for primary injury to axons in mild WMI is lacking, the deleterious effects of the abundant activated microglia and reactive astrocytes may disturb axonal development, separate from any effects on pre-OLs. In addition, as noted earlier, during normal development these glia are critical for axonal guidance and growth, and phenotypic diversion to activated or reactive cells could lead to dysmaturation. Thus a scenario similar to that depicted in Fig 9B seems possible.

      Thalamic, subplate, late migrating GABAergic neuron injury

      The scenarios described earlier for primary injury to these neural structures leading to dysmaturational gray matter disturbances in the setting of moderate to severe WMI (Fig 9C-E) cannot be ruled out in mild WMI but do not seem highly likely. For example, in the careful neuropathological study of Pierson et al.,
      • Pierson C.R.
      • Folkerth R.D.
      • Billards S.S.
      • et al.
      Gray matter injury associated with periventricular leukomalacia in the premature infant.
      in the 17 infants with DWMG (and no focal necroses), neuronal loss in cortex, thalamus, and basal ganglia was observed in none.

      Conclusions

      The dysmaturational disturbances of white matter and gray matter structures apparent by advanced MRI methods in infants with mild WMI do not appear to be related to widespread injury. Pre-OL injury and dysmaturation do seem apparent, and thus the possibility of the multiple secondary developmental disturbances of gray and white matter structures described earlier (Fig 9A) is real. The abundant reactive astrocytes and activated microglia in cerebral white matter, i.e., DWMG, are also likely important in the pre-OL injury or dysmaturation. In addition, axonal injury and dysmaturation are also a potential consequence of the deleterious actions of these two glial types (Fig 9B).

      Primary dysmaturation of gray matter structures

      The possibility that the gray matter structures shown to exhibit secondary impaired development with encephalopathy of prematurity, as outlined in the preceding discussion, may exhibit primary dysmaturation is suggested by recent clinical and experimental studies. If primary dysmaturation does occur, the approaches to neuroprotection and neurorestoration (see later) could be quite different from those directed at secondary dysmaturation in the context of cerebral WMI.

      Clinical data

      Primary dysmaturation of cerebral cortex, in the absence of evidence for WMI, is suggested by a study of 95 premature infants studied by MRI at two time points in the neonatal period (32 and 40 weeks post-conception).
      • Vinall J.
      • Grunau R.E.
      • Brant R.
      • et al.
      Slower postnatal growth is associated with delayed cerebral cortical maturation in preterm newborns.
      The principal finding was evidence for delayed microstructural development of cerebral cortical gray matter at multiple sites. Diffusion-based measurements showed delayed microstructural development in cerebral cortex, but not cerebral white matter, in association with impaired somatic growth. The expected normal developmental decline in FA in cortex was blunted, whereas the expected increase in FA in white matter was not. Thus no evidence for WMI or impaired white matter development could be identified. As described earlier,
      • Kinney H.C.
      • Volpe J.J.
      Organizational events.
      • Neil J.J.
      • Volpe J.J.
      Specialized neurological studies.
      FA decreases in the cortex principally with dendritic development. In the study of Vinall et al.
      • Vinall J.
      • Grunau R.E.
      • Brant R.
      • et al.
      Slower postnatal growth is associated with delayed cerebral cortical maturation in preterm newborns.
      radial diffusion, and not axial diffusion in the cortex, was particularly affected, again most consistent with impaired dendritic development. The association with impaired somatic growth raises the possibility that undernutrition is particularly involved, although detailed data regarding nutrition, caloric intake, and feeding were not available. However, it is noteworthy that several studies of premature newborns with intrauterine growth retardation also show a particular involvement of cerebral cortical development, including reduced cortical volume, reduced cortical surface area, and impaired gyrification.
      • Tolsa C.B.
      • Zimine S.
      • Warfield S.K.
      • et al.
      Early alteration of structural and functional brain development in premature infants born with intrauterine growth restriction.
      • Dubois J.
      • Benders M.
      • Borradori-Tolsa C.
      • et al.
      Primary cortical folding in the human newborn: an early marker of later functional development.
      • Lodygensky G.A.
      • Seghier M.L.
      • Warfield S.K.
      • et al.
      Intrauterine growth restriction affects the preterm infant's hippocampus.
      • Padilla N.
      • Falcon C.
      • Sanz-Cortes M.
      • et al.
      Differential effects of intrauterine growth restriction on brain structure and development in preterm infants: a magnetic resonance imaging study.
      • Xydis V.
      • Drougia A.
      • Giapros V.
      • et al.
      Brain growth in preterm infants is affected by the degree of growth restriction at birth.
      However, other studies of such infants have shown abnormalities in microstructural development of white matter.
      • Barnett M.L.
      • Tusor N.
      • Ball G.
      • et al.
      Exploring the multiple-hit hypothesis of preterm white matter damage using diffusion MRI.
      • Eikenes L.
      • Martinussen M.P.
      • Lund L.K.
      • et al.
      Being born small for gestational age reduces white matter integrity in adulthood: a prospective cohort study.
      Nonetheless, on balance, it does appear that disturbances in growth, perhaps secondary to undernutrition, either in the premature infant postnatally or in utero may have a primary dysmaturational effect on cerebral cortex. More data clearly are needed.

      Experimental data

      Three recent studies in a well-characterized preterm large animal (fetal sheep) model of cerebral ischemia raise the possibility of primary dysmaturation of cerebral cortex, subplate neurons, and caudate neurons.
      • Dean J.M.
      • McClendon E.
      • Hansen K.
      • et al.
      Prenatal cerebral ischemia disrupts MRI-defined cortical microstructure through disturbances in neuronal arborization.
      • McClendon E.
      • Chen K.
      • Gong X.
      • et al.
      Prenatal cerebral ischemia triggers dysmaturation of caudate projection neurons.
      • McClendon E.
      • Shaver D.C.
      • Degener-O'Brien K.
      • et al.
      Transient hypoxemia chronically disrupts maturation of preterm fetal ovine subplate neuron arborization and activitym.
      Thus utilizing elegant neurobiological methods, Back and coworkers have shown disturbances in cortex, in dendritic development and synapse formation; in subplate neurons, in dendritic arborization and synaptic activity; and in caudate, in dendritic arborization, synaptogenesis, and synaptic activity.
      • Dean J.M.
      • McClendon E.
      • Hansen K.
      • et al.
      Prenatal cerebral ischemia disrupts MRI-defined cortical microstructure through disturbances in neuronal arborization.
      • McClendon E.
      • Chen K.
      • Gong X.
      • et al.
      Prenatal cerebral ischemia triggers dysmaturation of caudate projection neurons.
      • McClendon E.
      • Shaver D.C.
      • Degener-O'Brien K.
      • et al.
      Transient hypoxemia chronically disrupts maturation of preterm fetal ovine subplate neuron arborization and activitym.
      These findings were apparent four weeks after the hypoxic-ischemic insult, but not after two weeks.
      • Dean J.M.
      • McClendon E.
      • Hansen K.
      • et al.
      Prenatal cerebral ischemia disrupts MRI-defined cortical microstructure through disturbances in neuronal arborization.
      As the basic experimental paradigm was designed originally to replicate cerebral WMI of the premature infant, these examples of neuronal dysmaturation were accompanied by pre-OL degeneration and dysmaturation and diffuse gliosis with reactive astrocytes and reactive microglia. A reasonable question is whether the four-week period required for the evolution of the cortical, subplate, or caudate neuronal dysmaturation is necessary because the dysmaturation is secondary to the pre-OL degeneration and dysmaturation as described earlier (Fig 9A). In the absence of a definitive answer to this question, the possibility that the hypoxic-ischemic insult leads primarily and directly to neuronal dysmaturations is real. Coupled with the clinical study described earlier, the latter possibility demands further research.

      Conclusions

      The clinical studies of premature infants with impaired somatic growth and of those with intrauterine growth retardation raise the possibility that cerebral cortical development may be affected directly, i.e., primarily, perhaps by nutritional factors. In view of the rapid development of cortex during the premature period and therefore its likely vulnerability to neonatal insults, such a possibility seems reasonable. Experimental data also raise the possibility of a primary dysmaturational effect for hypoxia-ischemia on cortical, subplate, and caudate neurons. However, as discussed, the available data do not rule out a primary effect on pre-OLs with secondary neuronal dysmaturation.

      Neuroprotective and neurorestorative interventions

      As the pervasive theme in this review is that pre-OL death leads to subsequent dysmaturation of both white and gray matter structures, interventions are best considered (1) as preventative of the initial death (i.e., neuroprotection) or (2) as amelioration or prevention of the subsequent dysmaturation (i.e., neurorestorative). Although there is overlap in this categorization, the distinction best facilitates the discussion that follows.

      Neuroprotective interventions

      Neuroprotective interventions have focused on prevention of pre-OL injury or death. Many excellent recent reviews have addressed this issue, and therefore this will not be discussed further.
      • Volpe J.J.
      • Kinney H.C.
      • Jensen F.E.
      • et al.
      The developing oligodendrocyte: key cellular target in brain injury in the premature infant.
      • Back S.A.
      • Volpe J.J.
      Encephalopathy of Prematurity: Pathophysiology.
      • Back S.A.
      White matter injury in the preterm infant: pathology and mechanisms.
      • Inder T.E.
      • Volpe J.J.
      Pathophysiology: General Principles.
      The principal neuroprotective interventions and the likely mechanism(s) affected in the cascade to pre-OL death are shown in Table 3. Most of the mechanisms are also relevant to those examples of WMI that are accompanied by direct injury to axons and neurons as well as to pre-OLs. Of the interventions shown in Table 3, only erythropoietin (EPO) has been studied in detail in human premature infants and will be discussed here.
      TABLE 3Neuroprotective Interventions to Prevent Pre-OL Injury/Death
      Mechanism TargetedInterventions
      Hypoxia-ischemia (perinatal or postnatal)Multiple
      Interventions related primarily to management of pregnancy, neonatal resuscitation, mechanical ventilation, bronchopulmonary dysplasia.
      Systemic Infection (perinatal or postnatal)Multiple
      Interventions related to prevention and treatment of fetal and neonatal systemic infection.
      Microglial Activation/InflammationMinocycline

      Melatonin
      ExcitotoxicityMemantine

      Topiramate
      Reactive oxygen or nitrogen species
       GenerationOxygenase/NOS inhibitors
       DefensesAntioxidative enzyme mimetics

      Free radical scavengers (N- acetylcysteine, vitamins E and K)
      Multiple mechanismsErythropoietin
      Studied in human preterm infants.
      ,
      Also promotes pre-OL differentiation.


      EGF
      Also promotes pre-OL differentiation.
      , IGF
      Also promotes pre-OL differentiation.


      Estradiol
      Abbreviations:
      EGF = Epidermal growth factor
      IGF = Insulin-like growth factor.
      MRI = Magnetic resonance imaging
      NOS = Nitric oxide synthase
      Interventions related primarily to management of pregnancy, neonatal resuscitation, mechanical ventilation, bronchopulmonary dysplasia.
      Interventions related to prevention and treatment of fetal and neonatal systemic infection.
      Studied in human preterm infants.
      § Also promotes pre-OL differentiation.

      Erythropoietin

      As EPO has antiexcitotoxic, antioxidant, antiinflammatory, and antiapoptotic effects,
      • Rangarajan V.
      • Juul S.E.
      Erythropoietin: emerging role of erythropoietin in neonatal neuroprotection.
      it is a prime candidate for the prevention of pre-OL injury or death, the critical initial event in genesis of preterm WMI. EPO has been shown to prevent or mitigate WMI in a variety of experimental models.
      • Rangarajan V.
      • Juul S.E.
      Erythropoietin: emerging role of erythropoietin in neonatal neuroprotection.
      • Rees S.
      • Hale N.
      • De Matteo R.
      • et al.
      Erythropoietin is neuroprotective in a preterm ovine model of endotoxin-induced brain injury.
      • Mazur M.
      • Miller R.H.
      • Robinson S.
      Postnatal erythropoietin treatment mitigates neural cell loss after systemic prenatal hypoxic-ischemic injury.
      Although numerous studies of EPO in premature infants have been carried out, a recent meta-analysis of four randomized controlled trials (RCTs) comprising 1133 infants is especially useful.
      • Fischer H.S.
      • Reibel N.J.
      • Buhrer C.
      • et al.
      Prophylactic early erythropoietin for neuroprotection in preterm infants: A meta-analysis.
      Prophylactic EPO administration reduced the incidence of Mental Developmental Index scores of less than 70 (odds ratio 0.51 [0.31 to 0.81], P < .005) at 18 to 24 months. As the total numbers of infants with less than 28 weeks' gestational age were not large enough to assess adequately the outcome in this critical group, more data are needed. A large multicenter randomized controlled trial in the United States (Preterm Erythropoietin Neuroprotection Trial, NCT01378273) is focused on this critical group, and results should be available this year.
      A closer assessment of the key EPO trials suggests that the timing of EPO administration may be critical in the likelihood of benefit. Thus, in one series of studies utilizing early, relatively brief administrations of EPO (atless than 3 hours, at 12 to 18 hours, and at 36 to 42 hours after birth), no significant differences in outcome at two years could be discerned
      • Natalucci G.
      • Latal B.
      • Koller B.
      • et al.
      Effect of early prophylactic high-dose recombinant human erythropoietin in very preterm enfants on neurodevelopmental outcome at 2 years: a randomized clinical trial.
      (although MRI at term equivalent age showed decreased WMI and better white matter maturation in the EPO-treated infants).
      • Leuchter R.H.
      • Gui L.
      • Poncet A.
      • et al.
      Association between early administration of high-dose erythropoietin in preterm infants and brain MRI abnormality at term-equivalent age.
      • O'Gorman R.L.
      • Bucher H.U.
      • Held U.
      • et al.
      Tract-based spatial statistics to assess the neuroprotective effect of early erythropoietin on white matter development in preterm infants.
      However, in a study utilizing EPO administration (as EPO or its higher glycosylated derivative darbepoetin) thrice weekly through 35 weeks' postconceptual age, the treated infants had better cognitive outcomes and less neurodevelopmental impairment at age 3.5 to four years, when compared with placebo-treated infants.
      • Ohls R.K.
      • Cannon D.C.
      • Phillips J.
      • et al.
      Preschool assessment of preterm infants treated with darbepoetin and erythropoietin.
      Thus the two different protocols with regard to the timing of EPO administration suggest that with the early, relatively brief approach, EPO was functioning only as a neuroprotective agent, whereas with the more prolonged approach the agent may have functioned both as a neuroprotective and a neurorestorative intervention. Perhaps consistent with this notion, the largest study to date randomized 800 infants of less than 32 weeks' gestation to placebo or EPO administered intravenously within 72 hours of birth and then once every other day for two weeks.
      • Song J.
      • Sun H.
      • Xu F.
      • et al.
      Recombinant human erythropoietin improves neurological outcomes in very preterm infants.
      The rate of moderate or severe neurological disability at 18 months' corrected age was significantly lower in the EPO group (7.1%) versus the placebo group (18.8%) (odds ratio = 0.22, confidence interval, 0.19 to 0.55, P < .001). Dosing in the aforementioned Preterm Erythropoietin Neuroprotection Trial will be still more prolonged, i.e., initially, single doses intravenously, every other day, from day one to day 11, and subsequently, doses subcutaneously every other day until 32 weeks. The potential mechanisms for EPO's benefit concerning brain maturation, i.e., neurorestorative effects, will be discussed in the next section.

      Neurorestorative Interventions

      The emphasis of this review has been on the evolution of the widespread dysmaturational events that follow the initial insult(s) and injury or death, especially to pre-OLs. These events develop over many weeks to months, and perhaps longer. This relatively protracted period raises the possibility of a long time window for interventions potentially capable of ameliorating or preventing the dysmaturation. I will term these interventions neurorestorative. The principal such interventions, shown in Tables 4 and 5, are classified based on their study in experimental settings only (Table 4) or in clinical settings with human infants, principally preterm infants (Table 5).
      TABLE 4Neurorestorative Interventions - Experimental Studies
      Major Interventions
       EGF, IGF-1
       Hyaluronidase inhibitors
       Microglial or astrocytic manipulation
       Stem cells
       Exosomes
       Dendrimers
      Abbreviations:
      EGF = Epidermal growth factor
      IGF-1 = Insulin-like growth factor
      TABLE 5Neurorestorative Interventions - Clinical Studies
      Major Interventions
       Erythropoietin
       Nutritional factors
      Quality and source of milk
      Components of milk
      Breastfeeding
      Polyunsaturated fatty acids
      Iron
      Zinc
       Experiential factors
      Neonatal period
      Auditory
      Visual
      Pain, stress
      Post-term
      Early intervention programs
      Parenting, educational, or social factors

      Experimental studies

      EGF and IGF-1

      Both epidermal growth factor (EGF) and insulinlike growth factor (IGF-1) have beneficial effects in experimental models of preterm WMI (Table 4). The agents appear to exhibit both neuroprotective and neurorestorative properties. In a mouse model of preterm WMI, Scafidi et al. showed that either selective overexpression of human EGF receptor in OL lineage cells or the intranasal administration of EGF immediately after injury led to decreased OL death, enhanced generation of new OLs from progenitor cells, and promoted functional recovery.
      • Scafidi J.
      • Hammond T.R.
      • Scafidi S.
      • et al.
      Intranasal epidermal growth factor treatment rescues neonatal brain injury.
      The benign mode of administration of the EGF suggests potential clinical applicability.
      IGF-1 has shown protective effects versus WMI both in neonatal animal models (hypoxia-ischemia, lipopolysaccharide-induced inflammation) and in cultured pre-OLs.
      • Cao Y.
      • Gunn A.J.
      • Bennet L.
      • et al.
      Insulin-like growth factor (IGF)-1 suppresses oligodendrocyte caspase-3 activation and increases glial proliferation after ischemia in near-term fetal sheep.
      • Brywe K.G.
      • Mallard C.
      • Gustavsson M.
      • et al.
      IGF-I neuroprotection in the immature brain after hypoxia-ischemia, involvement of Akt and GSK3beta?.
      • Wood T.L.
      • Loladze V.
      • Altieri S.
      • et al.
      Delayed IGF-1 administration rescues oligodendrocyte progenitors from glutamate-induced cell death and hypoxic-ischemic brain damage.
      • Zhong J.
      • Zhao L.
      • Du Y.
      • et al.
      Delayed IGF-1 treatment reduced long-term hypoxia-ischemia-induced brain damage and improved behavior recovery of immature rats.
      • Pang Y.
      • Zheng B.
      • Campbell L.R.
      • et al.
      IGF-1 can either protect against or increase LPS-induced damage in the developing rat brain.
      The agent also showed restorative effects, i.e., rescue of pre-OLs and promotion of myelination. Two issues limit enthusiasm for IGF-1: first, the peptide must be administered intraventricularly, and second, its effects are dose-related, with lower doses being effective but higher doses being toxic.

      Hyaluronidase inhibitors

      Pre-OL dysmaturation in chronic WMI appears related at least in considerable part to the astrocytic component of the diffuse gliosis characteristic of the lesion. Thus Back and coworkers have shown that reactive astrocytes synthesize high-molecular-weight forms of hyaluronic acid, which are readily detectable in the human lesion.
      • Buser J.R.
      • Maire J.
      • Riddle A.
      • et al.
      Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants.
      • Back S.A.
      White matter injury in the preterm infant: pathology and mechanisms.
      As described earlier, hyaluronic acid digestion products, generated from hyaluronidases in the disrupted ECM of WMI, lead to a block in pre-OL maturation. This block could be prevented by pharmacologic inhibition of hyaluronidase in vitro and in an animal model.
      • Preston M.
      • Gong X.
      • Su W.
      • et al.
      Digestion products of the PH20 hyaluronidase inhibit remyelination.
      Whether use of a hyaluronidase inhibitor has value in preventing pre-OL dysmaturation in the human infant requires further study.

      Microglial or astrocytic phenotypic manipulation

      Abundant microglia are important components of the diffuse gliotic component of WMI (see earlier). These cells are principally in an activated, pro-inflammatory state (M1 phenotype). Their role in acute pre-OL injury or death likely relates to the generation of reactive oxygen and nitrogen species and secretion of injurious cytokines.
      • Volpe J.J.
      • Kinney H.C.
      • Jensen F.E.
      • et al.
      The developing oligodendrocyte: key cellular target in brain injury in the premature infant.
      However, a variety of studies, performed in in vitro and in vivo models, including adult human lesions with failure of OL differentiation and myelin development (e.g., multiple sclerosis), suggest involvement of activated microglia in the subsequent pre-OL dysmaturation in human preterm WMI.
      • Favrais G.
      • van de Looij Y.
      • Fleiss B.
      • et al.
      Systemic inflammation disrupts the developmental program of the white matter.
      • Miron V.E.
      • Boyd A.
      • Zhao J.W.
      • et al.
      M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination.
      • Krishnan M.L.
      • Van Steenwinckel J.
      • Schang A.L.
      • et al.
      Integrative genomics of microglia implicates DLG4 (PSD95) in the white matter development of preterm infants.
      The data raise the possibility that interventions capable of converting microglia from a pro-inflammatory phenotype (M1) to an anti-inflammatory phenotype (M2) would have major potential as a neurorestorative therapy. Such immunomodulatory agents that cross the blood-brain barrier have been identified (e.g., minocycline, melatonin, minozac, etanercept) and are under study in human adult demyelinating diseases.
      • Donat C.K.
      • Scott G.
      • Gentleman S.M.
      • et al.
      Microglial activation in traumatic brain injury.
      • Miron V.E.
      • Boyd A.
      • Zhao J.W.
      • et al.
      M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination.
      • Michell-Robinson M.A.
      • Touil H.
      • Healy L.M.
      • et al.
      Roles of microglia in brain development, tissue maintenance and repair.
      Their safety and efficacy in the premature infant for long-term use are not established.
      • Biran V.
      • Phan Duy A.
      • Decobert F.
      • et al.
      Is melatonin ready to be used in preterm infants as a neuroprotectant?.
      A recent relevant area of interest in the regulation of microglial phenotype involves microRNAs.
      • Michell-Robinson M.A.
      • Touil H.
      • Healy L.M.
      • et al.
      Roles of microglia in brain development, tissue maintenance and repair.
      • Ksiazek-Winiarek D.
      • Szpakowski P.
      • Turniak M.
      • et al.
      IL-17 exerts anti-apoptotic affect via miR-155-5p downregulation in experimental autoimmune encephalomyelitis.
      These components are short noncoding RNAs (18 to 22 nucleotides), which are transcriptional regulators of gene expression. Several microRNAs have been shown to promote or inhibit inflammatory responses in microglia. One prominent microRNA of activated microglia is mir-155, which is elevated in microglia in multiple sclerosis lesions. When silenced in vivo by intracerebral injection of IL-17 in early stages of experimental allergic encephalomyelitis, the pathologic and clinical effects of the demyelinating disease are blunted.
      • Ksiazek-Winiarek D.
      • Szpakowski P.
      • Turniak M.
      • et al.
      IL-17 exerts anti-apoptotic affect via miR-155-5p downregulation in experimental autoimmune encephalomyelitis.
      Thus the possibility of such systemic therapy seems real. Indeed, recent research shows that intravenous delivery of another microRNA (miR-124) that promotes polarization of microglia from an inflammatory (M1) to an anti-inflammatory (M2) phenotype via miR-124-enriched exosomes improves hippocampal neurogenesis and neurological function over four weeks after traumatic brain injury
      • Yang Y.
      • Ye Y.
      • Kong C.
      • et al.
      MiR-124 Enriched exosomes promoted the M2 polarization of microglia and enhanced hippocampus neurogenesis after traumatic brain injury by inhibiting TLR4 pathway.
      (see also later discussion of exosomes). Notably, because the anti-inflammatory phenotype of microglia (M2) is important in the facilitation of many brain developmental events as described earlier, these avenues of research suggest a major neurorestorative possibility for in vivo manipulation of microglia phenotype.
      Similar considerations concerning glial manipulation from a “harmful” to developmentally “helpful” phenotype apply to the reactive astrocytes in the diffuse gliotic component. Their involvement in pre-OL injury and dysmaturation and the potential value of hyaluronidase inhibitors were discussed earlier. Prevention of the microglial induction of harmful, “neurotoxic” astrocytes (A1 phenotype) is an area of active current research.
      • Liddelow S.A.
      • Barres B.A.
      Reactive Astrocytes: Production, Function, and Therapeutic Potential.
      • Liddelow S.A.
      • Guttenplan K.A.
      • Clarke L.E.
      • et al.
      Neurotoxic reactive astrocytes are induced by activated microglia.
      The valuable results would be inhibition of pre-OL death and preservation of the maturational effects of the developmentally beneficial astrocytic phenotype (A2). A variety of drugs and neutralizing antibodies (e.g., to tumor necrosis factor-α and IL-α from microglia) to prevent induction of harmful reactive astrocytes are under study in animal models and in adult human neurodegenerative disorders.
      • Liddelow S.A.
      • Barres B.A.
      Reactive Astrocytes: Production, Function, and Therapeutic Potential.

      Stem cells

      Experimental studies of stroke and related ischemic brain injuries in neonatal animals suggest that stem cell therapies may be effective for restoration, particularly of OLs.
      • Titomanlio L.
      • Kavelaars A.
      • Dalous J.
      • et al.
      Stem cell therapy for neonatal brain injury: perspectives and challenges.
      • van Velthoven C.T.
      • Sheldon R.A.
      • Kavelaars A.
      • et al.
      Mesenchymal stem cell transplantation attenuates brain injury after neonatal stroke.
      • Fleiss B.
      • Guillot P.V.
      • Titomanlio L.
      • et al.
      Stem cell therapy for neonatal brain injury.
      • van Velthoven C.T.
      • Gonzalez F.
      • Vexler Z.S.
      • et al.
      Stem cells for neonatal stroke- the future is here.
      • Wei Z.Z.
      • Gu X.
      • Ferdinand A.
      • et al.
      Intranasal delivery of bone marrow mesenchymal stem cells improved neurovascular regeneration and rescued neuropsychiatric deficits after neonatal stroke in rats.
      • Li J.
      • Yawno T.
      • Sutherland A.
      • et al.
      Term vs. preterm cord blood cells for the prevention of preterm brain injury.
      The major types of cells used thus far include neural, embryonic, mesenchymal, umbilical cord, and induced pluripotent cells. In vitro manipulation of neural precursor cells before transplantation can enhance their capacity to undergo OL differentiation and axonal remyelination.
      • Nagoshi N.
      • Khazaei M.
      • Ahlfors J.E.
      • et al.
      Human spinal oligodendrogenic neural progenitor cells promote functional recovery after spinal cord injury by axonal remyelination and tissue sparing.
      A variety of routes of cell administration have been utilized, and intranasal administration may be the most efficient. Stem cells administered by this route appear to target the injury site after entering the brain via olfactory neural processes traversing the cribriform plate.
      • Li Y.H.
      • Feng L.
      • Zhang G.X.
      • et al.
      Intranasal delivery of stem cells as therapy for central nervous system disease.
      Studies of rodent models of preterm brain injury have shown that the intranasal route of administration can be effective not only for mitigating injury to myelin but also for improving behavioral outcome.
      • van Velthoven C.T.
      • Sheldon R.A.
      • Kavelaars A.
      • et al.
      Mesenchymal stem cell transplantation attenuates brain injury after neonatal stroke.
      • Oppliger B.
      • Joerger-Messerli M.
      • Mueller M.
      • et al.
      Intranasal delivery of umbilical cord-derived mesenchymal stem cells preserves myelination in perinatal brain damage.
      Of particular relevance to cerebral WMI and pre-OL dysmaturation or myelination failure in the premature infant is a recent study of such injury produced by hypoxia-ischemia in the 3-day-old rat.
      • Chen L.X.
      • Ma S.M.
      • Zhang P.
      • et al.
      Neuroprotective effects of oligodendrocyte progenitor cell transplantation in premature rat brain following hypoxic-ischemic injury.
      OL progenitor cells produced from embryonic stem cells were transplanted into the injured cerebrum. The transplanted cells survived, underwent differentiation, formed myelin sheaths, and stimulated proliferation of endogenous neural stem cells. Supporting a neurorestorative effect was the demonstration of functional benefit after six weeks. It will be of particular interest if the results can be replicated after intranasal administration.
      A relevant human study in this context involves the transplantation of human neural stem cells into the brains of four infants with connatal Pelizaeus-Merzbacher disease.
      • Osorio M.J.
      • Rowitch D.H.
      • Tesar P.
      • et al.
      Concise review: Stem cell-based treatment of Pelizaeus-Merzbacher disease.
      After one year, evidence of myelin ensheathment of axons was obtained by diffusion tensor imaging. Direct extrapolation to the human preterm infant with WMI is difficult because of the mode of administration used. Nevertheless, the findings suggest promise for stem cell therapy as a neurorestorative therapy in such infants.

      Exosomes

      The precise neuroprotective factors released by stem cells are not known with certainty and may vary as a function of the injury. Notably, however, effects on pre-OL and myelin development and on behavioral outcome in a rodent model of preterm WMI was achieved with intravenous administration of extracellular vesicles, i.e., exosomes, derived from stem cells, in lieu of stem cells per se.
      • Drommelschmidt K.
      • Serdar M.
      • Bendix I.
      • et al.
      Mesenchymal stem cell-derived extracellular vesicles ameliorate inflammation-induced preterm brain injury.
      (Exosomes are a type of extracellular vesicle and can carry membrane and cytosolic proteins, various types of RNA and lipids, and perhaps DNA).
      • Holm M.M.
      • Kaiser J.R.
      • Schwab M.E.
      Extracellular vesicles: Multimodal envoys in neural maintenance and repair.
      Similar benefit from the use of exosomes has been demonstrated in other animal models of brain injury.
      • Otero-Ortega L.
      • Gomez de Frutos M.C.
      • Laso-Garcia F.
      • et al.
      Exosomes promote restoration after an experimental animal model of intracerebral hemorrhage.
      • Williams A.
      • Dennahy I.S.
      • Bhatti U.F.
      • et al.
      Mesenchymal stem cell-derived exosomes provide neuroprotection and improve long-term neurologic outcomes in a swine model of traumatic brain injury and hemorrhagic shock.
      • Zhao L.
      • Jiang X.
      • Shi J.
      • et al.
      Exosomes derived from bone marrow mesenchymal stem cells overexpressing microRNA-25 protect spinal cords against transient ischemia.
      The great therapeutic potential of exosomes, isolated from blood, has been recognized only recently, and the capacity to induce OL differentiation and myelination could serve a crucial neurorestorative function in the premature infant. Human studies will be of great interest.

      Dendrimers

      Dendrimers are unique nanoparticles synthesized for a variety of functions, including targeted delivery of therapeutic agents to brain.
      • Menjoge A.R.
      • Kannan R.M.
      • Tomalia D.A.
      Dendrimer-based drug and imaging conjugates: design considerations for nanomedical applications.
      • Kannan R.M.
      • Nance E.
      • Kannan S.
      • et al.
      Emerging concepts in dendrimer-based nanomedicine: from design principles to clinical applications.
      Their small size and tailorable surface functional groups make them valuable for this role. Drugs, and perhaps ultimately, microRNAs or silencing RNAs can be attached to the dendrimer. Several recent models of ischemia- or inflammation-induced neonatal or fetal brain injury have shown marked beneficial effects of dendrimer–N-acetylcysteine conjugates.
      • Kannan S.
      • Dai H.
      • Navath R.S.
      • et al.
      Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model.
      • Kim I.D.
      • Shin J.H.
      • Kim S.W.
      • et al.
      Intranasal delivery of HMGB1 siRNA confers target gene knockdown and robust neuroprotection in the postischemic brain.
      • Nance E.
      • Porambo M.
      • Zhang F.
      • et al.
      Systemic dendrimer-drug treatment of ischemia-induced neonatal white matter injury.
      • Nemeth C.L.
      • Drummond G.T.
      • Mishra M.K.
      • et al.
      Uptake of dendrimer-drug by different cell types in the hippocampus after hypoxic-ischemic insult in neonatal mice: Effects of injury, microglial activation and hypothermia.
      • Lei J.
      • Rosenzweig J.M.
      • Mishra M.K.
      • et al.
      Maternal dendrimer-based therapy for inflammation-induced preterm birth and perinatal brain injury.
      N-acetylcysteine is an antioxidant, and after intravenous administration of the conjugate, uptake into activated microglia, reactive astrocytes, and differentiating OLs could be demonstrated. Sustained prevention of OL injury and improved myelination were shown.
      • Nance E.
      • Porambo M.
      • Zhang F.
      • et al.
      Systemic dendrimer-drug treatment of ischemia-induced neonatal white matter injury.
      The principal cellular target appeared to be inflammatory microglia. Further studies will be of great interest.

      Clinical studies

      A burgeoning clinical literature suggests the possibility that the dysmaturation of both pre-OLs and gray matter structures after premature brain injury can be counteracted to a considerable extent. These neurorestorative interventions include pharmacologic agents, i.e., EPO, and modifications of nutrition and other environmental factors (Table 5). Implementation of these interventions during the vulnerable neonatal period, when the remarkable array of developmental events described earlier are proceeding most rapidly, is of critical importance. However, the beneficial effects of these interventions likely continue beyond the neonatal period (see later). The mechanisms of the benefits and the specific maturational events affected are not yet entirely understood. Our current understanding of these interventions is discussed next.

      Erythropoietin

      EPO was discussed earlier in relation to its neuroprotective effects. Notably as discussed earlier, current data suggest that more prolonged exposure to EPO is more beneficial than only early and brief exposure, thus suggesting that EPO has neurorestorative as well as neuroprotective properties.
      Experimental data suggest that neurorestorative effects of EPO involve particularly OL development, although promotion of angiogenesis and neurogenesis may also occur.
      • Neil J.J.
      • Volpe J.J.
      Encephalopathy of prematurity: Clinical-neurological features, diagnosis, imaging, prognosis, therapy.
      • Rangarajan V.
      • Juul S.E.
      Erythropoietin: emerging role of erythropoietin in neonatal neuroprotection.
      • Weisglas-Kuperus N.
      • Heersema D.J.
      • Baerts W.
      • et al.
      Visual functions in relation with neonatal cerebral ultrasound, neurology and cognitive development in very-low-birthweight children.
      In view of the likely importance of the failure of differentiation of pre-OLs in the genesis of axonal and neuronal dysmaturation, the decisive role of EPO in promoting pre-OL development after hypoxic-ischemic insults in experimental models is particularly relevant here.
      • Iwai M.
      • Stetler R.A.
      • Xing J.
      • et al.
      Enhanced oligodendrogenesis and recovery of neurological function by erythropoietin after neonatal hypoxic/ischemic brain injury.
      • Kato S.
      • Aoyama M.
      • Kakita H.
      • et al.
      Endogenous erythropoietin from astrocyte protects the oligodendrocyte precursor cell against hypoxic and reoxygenation injury.
      • Jantzie L.L.
      • Miller R.H.
      • Robinson S.
      Erythropoietin signaling promotes oligodendrocyte development following prenatal systemic hypoxic-ischemic brain injury.
      In vivo, EPO appears to be generated primarily from astrocytes, abundantly present in the diffuse gliotic component of WMI. However, the EPO receptor is upregulated in pre-OLs after hypoxic-ischemic insults, and if sufficient endogenous EPO is not present, the unoccupied receptor leads to a failure of differentiation. The provision of abundant exogenous EPO may explain the benefit of EPO therapy vis-à-vis pre-OL differentiation. An indirect effect of EPO on pre-OL and neuronal or axonal maturation may also relate to its action of decreasing microglial recruitment, the other key glial element in DWMG, and thereby the deleterious effects of inflammation.
      • Liu W.
      • Shen Y.
      • Plane J.M.
      • et al.
      Neuroprotective potential of erythropoietin and its derivative carbamylated erythropoietin in periventricular leukomalacia.
      In addition, in an animal model,
      • Jantzie L.L.
      • Corbett C.J.
      • Firl D.J.
      • et al.
      Postnatal erythropoietin mitigates impaired cerebral cortical development following subplate loss from prenatal hypoxia-ischemia.
      EPO also promotes cerebral cortical development after hypoxia-ischemia and associated subplate neuronal loss, again consistent with its multifaceted effects on cellular development in brain. Recall that in moderate to severe WMI in premature infants, subplate neuronal loss and impaired cortical development are important features (see earlier).
      A major question regarding EPO as a restorative therapy relates to the prolonged duration likely required. Reactive astrocytes and activated microglia are likely present for many months after the initial injury. More data are needed regarding the safety of such prolonged treatment with an agent with multifaceted developmental effects.

      Nutritional factors

      The importance of appropriate nutrition during the premature period for neurodevelopmental outcome and the deleterious effects of postnatal undernutrition are supported by a large corpus of clinical, epidemiologic, and experimental studies.
      • Hayakawa M.
      • Okumura A.
      • Hayakawa F.
      • et al.
      Nutritional state and growth and functional maturation of the brain in extremely low birth weight infants.
      • Latal-Hajnal B.
      • Von Siebenthal K.
      • Kovari H.
      • et al.
      Postnatal growth in VLBW infants: significant association with neurodevelopmental outcome.
      • Ehrenkranz R.A.
      • Dusick A.M.
      • Vohr B.R.
      • et al.
      Growth in the neonatal intensive care unit influences neurodevelopmental and growth outcomes of extremely low birth weight infants.
      • Lira P.I.C.
      • Eickmann S.E.
      • Lima M.C.
      • et al.
      Early head growth: relation with IQ at 8 years and determinants in term infants of low and appropriate birthweight.