Abstract
The major neuropathological substrate of human preterm brain injury is the encephalopathy of prematurity, a term coined to characterize the multifaceted gray and white matter lesions in the preterm brain that reflect acquired insults, altered developmental trajectories, and reparative phenomena in various combinations. Because the responsible insults occur at a time of rapid brain growth, a host of developmental programs may be affected, resulting in maturational defects that compound the acquired lesion (e.g., hypoxic-ischemic injury leading to loss of preoligodendrocytes [pre-OLs] and impaired myelination). The cause of the encephalopathy of prematurity is multifactorial and includes cerebral hypoxia-ischemia and systemic infection/inflammation, resulting in glutamate, free radical, and/or cytokine toxicity to pre-OLs, axons, and neurons. Cerebral white matter injury represents a spectrum of disease by neuropathological study, with “focal necrotic/cystic” periventricular leukomalacia (PVL) the most striking and diffuse “nonnecrotic/noncystic disease” the least striking. Nonnecrotic/noncystic cerebral white matter injury, a more diffuse abnormality, in which cysts or focal necrotic lesions are either not present or are minute in size and below the level of resolution of conventional neuroimaging, accounts for the majority of white matter disease in modern neonatal intensive care units. Magnetic resonance imaging (MRI) is the most effective imaging modality for the detection of this milder form of cerebral white matter disease and shows diffuse excessive high signal intensity in cerebral white matter, possibly reflecting diffuse gliosis. The patterns and mechanisms of injury are highly dependent on the specific maturational stages of OLs, neurons, and axons over the last half of gestation (i.e., the time frame of occurrence of the encephalopathy).
Keywords
encephalopathy of prematurity, diffuse gliosis, periventricular leukomalacia, preoligodendrocytes, axonopathy, gray matter injury
The major neuropathological substrate of human preterm brain injury is the encephalopathy of prematurity , a term coined to characterize the multifaceted gray and white matter lesions in the preterm brain that reflect acquired and developmental factors in combination ( Table 14.1 ). The encephalopathy of prematurity is also associated with hemorrhages, notably in the germinal matrix of the ganglionic eminence (GE) (see Chapter 24 ) and cerebellum (see Chapter 23 ) and with focal micro- or macroinfarcts. Because the responsible insults occur at a time of rapid brain growth, a host of developmental programs may be affected, resulting in maturational defects that compound the acquired lesion (e.g., hypoxic-ischemic injury leading to loss of preoligodendrocytes [pre-OLs] and subsequent impaired maturation and, as a consequence, impaired myelination). The cause of the encephalopathy of prematurity is multifactorial and includes cerebral hypoxia-ischemia and systemic infection/inflammation, which results in glutamate, free radical, and/or cytokine toxicity to pre-OLs, axons, and neurons (see Chapter 15 ). Contributory roles for impaired nutrition, pain, stress, drugs and other factors associated with neonatal intensive care seem likely but remain to be clarified (see Chapters 15 and 16 ). Given the heterogeneity and diverse combinations of the lesions that comprise the encephalopathy of prematurity, it is not surprising that the spectrum of neurodevelopmental abnormalities in preterm survivors is wide and includes, often in combination, a variety of cognitive, behavioral, socialization, attentional, and motor deficits (see Chapter 16 ).The patterns and mechanisms of injury are highly dependent upon the specific maturational stages of OLs, neurons, and axons over the last half of gestation (i.e., the time frame of occurrence of the encephalopathy).
|
Neuropathological Features of the Encephalopathy of Prematurity
Periventricular Leukomalacia
The central feature of the encephalopathy of prematurity is periventricular leukomalacia (PVL). This lesion is the major white matter component of the encephalopathy and is defined as focal periventricular necrosis associated with more diffuse reactive gliosis and microglial activation in the surrounding cerebral white matter. Thus PVL has two components, focal and diffuse. The seminal publication of Banker and Larroche in 1962 characterized this lesion in detail and related it to adverse cardiorespiratory events and cerebral ischemia in the affected infants, with the focal lesion apparently occurring in deep arterial border zones and end zones in the periventricular white matter ( Fig. 14.1 ).
The necrotic foci likely represent a core infarct with destruction of all cellular elements, while the astrocytic and microglial response in the surrounding white matter represents less severe and potentially reversible ischemic injury. The necrotic foci progress from coagulative necrosis (characteristic of the histology of tissue ischemia in all tissues, with hypereosinophilia, nuclear pyknosis, and axonal spheroids) to organizing necrosis with reactive gliosis, macrophagocytic infiltration, and tissue disintegration and then end-stage cystic formation and gliosis ( Fig. 14.2 ). This latter variety of PVL (i.e., with focal cysts, the more severe end of a spectrum) is termed cystic PVL in the neuroimaging literature (see the section on neuropathology in living infants , later).
Importantly, the necrotic foci are not always apparent upon macroscopic examination . In autopsy studies in our hospital from the modern era of intensive care, 46% to 82% of PVL cases, depending upon the data set, have only microscopic necrotic foci (with macrophagocytic infiltration) that measure less than 2 mm in diameter ( Fig. 14.3 ). These punctate white matter lesions are now the most common focal manifestation of PVL at autopsy.
Nevertheless, visually obvious foci of necrosis, or “white spots,” as well as cysts greater than 2 mm in diameter are still detected at autopsy ( Fig. 14.4 ). The small necrotic foci do not result in cysts but rather focal areas of gliosis. In the neuroimaging literature, this variety of PVL is often termed noncystic PVL . The small foci of gliosis may account for the focal punctate lesions seen by magnetic resonance imaging (MRI) (see the section on neuropathology in living infants , later, and Chapter 16 ), although careful anatomical imaging correlations are lacking.
Diffuse white matter gliosis without periventricular necrotic foci occurs frequently in preterm brains, but its relationship to PVL is uncertain (e.g., whether or not it represents the least severe end of a spectrum of ischemic injury to the premyelinated white matter, with PVL at the most severe end). Reactive gliosis and activated microglia are the two major inflammatory components of PVL ( Fig. 14.5 ). Presumed to be initially protective against pre-OL cell damage, these cells carry the potential for compounding tissue injury when the insult is prolonged and/or severe. Reactive gliosis in PVL is preferentially located in the deep as opposed the intragyral white matter and thereby may define injury in the vascular distal fields of the cerebral white matter. Activated microglia likewise conform to this regional distribution, while macrophages are prominent in the organizing necrotic foci of the periventricular regions. Both astrocytes and microglia/macrophages produce inflammatory cytokines, and immunocytochemical studies in PVL demonstrate increased cytokine expression within them as a distinctive feature of the histopathology. Notably, reactive astrocytes in PVL express interferon-γ and thus are a potential source for this toxic cytokine, particularly to pre-OLs compared with mature OLs. Reactive astrocytes and microglia/macrophages also help to protect pre-OLs from excitotoxic injury by the upregulation of the glutamate transporter excitatory amino acid transporter (EAAT) and uptake of excessive extracellular glutamate, as suggested by the finding that the percentage of EAAT2-immunopositive astrocytes is increased in PVL as compared with control white matter; moreover, macrophages in the necrotic foci express EAAT2. Yet reactive astrocytes and microglia may contribute to free radical injury in PVL, as indicated by intense expression of inducible nitric oxide synthase (iNOS), a marker of nitrative stress, in reactive astrocytes in the acute through chronic stages of PVL, and in activated microglia primarily in the acute stage, this last observation suggesting an early role for microglial iNOS in the pathogenesis of PVL. In addition, the density of iNOS-immunopositive cells is significantly increased in the diffuse component in cerebral white matter.
The cellular evolution of PVL involves acute loss of pre-OLs ; some OL cell bodies appear to survive with loss of cell processes, others with morphological dysfunction in myelin formation as well as subsequent hypomyelination. Immunocytochemical analysis using an antibody to Olig2, a pan-OL lineage marker, indicates no significant difference in Olig2 cell density in the periventricular or intragyral white matter between PVL cases and controls. Moreover, early lineage markers suggest that there is an attempt at replenishment of pre-OLs by proliferation of progenitors, but these pre-OLs fail to mature. The cellular result is dominance of pre-OLs over mature OLs (see further on and Chapter 15 ). Consistent with a maturational disturbance of pre-OLs, qualitative abnormalities of myelin basic protein (MBP) staining in both the diffuse and necrotic components of PVL occur despite preserved Olig2 cell density. These include excessive MBP immunostaining in enlarged OL perikarya that presumably reflects a functional derangement in MBP transport from its site of production in the OL cell body to the OL processes.
Free radical injury to pre-OLs in PVL is indicated by early immunocytochemical evidence for protein nitration and lipid peroxidation of pre-OLs in the diffusely gliotic component of PVL ( Fig. 14.6 ). In addition, F(2)-isoprostanes, an arachidinate metabolite/lipid peroxidation marker of oxidative damage, is significantly increased in the white matter of early PVL cases. The end stage of PVL is delayed myelination or hypomyelination of the cerebral white matter and compensatory ventricular enlargement.
Gray Matter Lesions in Encephalopathy of Prematurity
Neuronal loss and/or gliosis are the histopathological hallmarks of gray matter injury in the encephalopathy of prematurity and occur in virtually all gray matter sites, albeit in variable combinations ( Fig. 14.7 ). Over one-third of PVL cases demonstrate overt gray matter lesions characterized by neuronal loss and/or gliosis ; microglial activation is often striking. Of note, more refined techniques, such as analysis of dendritic and spine number and morphology, may ultimately detect neuronal deficits at the subcellular (and molecular) levels (see Chapter 15 ). The incidence of neuronal loss, as assessed semiquantitatively in tissue sections, is 38% in the thalamus, 33% in the globus pallidus and hippocampus, and 29% in the cerebellar dentate nucleus. Gliosis without obvious neuronal loss is more common than combined neuronal loss and gliosis, occurring in the thalamus (56% of PVL cases), globus pallidus (60%), hippocampus (47%), basis pontis (100%), inferior olive (92%), and brain-stem tegmentum (43%). In a histopathological survey of brain injury in infants with very low birth weights, the frequency of neuronal loss (sites unspecified) is reportedly less than that of cerebral white matter abnormalities. Because detection of neuronal loss and gliosis is a somewhat crude measure of neuronal disturbance, the possibility of even more frequent neuronal disturbance is likely. Moreover, neuronal loss and gliosis may not reflect primary injury but rather secondary dysmaturational effects caused by trophic and retrograde and anterograde disturbances (see later).
Thalamus
Thalamic injury associated with PVL may be heterogeneous and occur in different patterns, reflecting different types of insults. Four different patterns of thalamic injury have been recognized: (1) diffuse gliosis with or without neuronal loss; (2) microinfarcts with focal neuronal loss; (3) macroinfarcts in the distribution of the posterior cerebral artery; and (4) status marmoratous. These different patterns likely reflect separate pathogenetic mechanisms, including diffuse hypoxia-ischemia and focal arterial embolism, as well as potentially different temporal characteristics of the responsible insults. Diffuse diminutions in thalamic volume are seen in older children with PVL ( Fig. 14.8 ). The thalamic volumetric disturbances could reflect either direct injury or secondary anterograde and retrograde effects related to axonal and subplate neuron disturbance (see later).
Cerebral Cortex
During the last half of gestation, the neocortex transforms from an undifferentiated cortical plate to a highly specialized structure (see Chapter 7 ). Around 30 gestational weeks, the cortical plate comprises six layers, each of which is characterized by a specific composite of differentiating pyramidal and nonpyramidal neurons. The cortex increases in thickness because of striking increases in the neuropil (e.g., neuronal cell size, dendritic arborization, spine formation, and arrival of preterminal afferents) (see Chapter 7 ). Relative to excitotoxicity, the excitatory amino acid receptor GluR2 is low in the pyramidal and nonpyramidal neurons in the cerebral cortex during the preterm period. In a study of PVL cases compared to controls adjusted for postconceptional age, there was a marked reduction (38%) in the density of layer V neurons in all areas sampled in the PVL cases ( n = 17) compared with controls ( n = 12) adjusted for postconceptional age at or greater than 30 weeks, when the six-layer cortex is visually distinct ( P < .024). This reduction may reflect a dying-back loss of somata secondary to transection of layer V axons projecting through the necrosis in the underlying white matter. This study underscores the role of secondary cortical effects in the encephalopathy of prematurity.
Late Migrating GABAergic Neurons
A defining feature of cortical development in the human preterm period is the late development of the GABAergic interneurons that play a key role in cortical specification, output, and synaptic plasticity (see Chapter 7 ). At least 20% of GABAergic neurons migrate through the white matter to the cerebral cortex over late gestation. This migration peaks around term and then declines and ends within the first 6 postnatal months; in parallel, the GABAergic neuronal density increases in the cortex over late gestation, peaks at term, and declines thereafter. One report has shown a deficit in GABAergic neurons in cerebral white matter in infants with PVL.
Subplate Neurons
Deficit of Neurons in the Subplate Zone and White Matter in the Encephalopathy of Prematurity.
There is damage to neurons not only in gray matter sites but also to those located in the cerebral white matter and subplate region. The density of granular neurons is significantly reduced in the periventricular and central white matter and the subplate region in PVL. These neurons are likely late-migrating GABAergic neurons and/or non-GABAergic constituents of the subplate region and interstitial white matter. In regard to the former possibility, a reduction in the density of GAD67-immunopositive neurons and neurons expressing the GABA A α1 receptor has been reported in human perinatal white matter lesions (with and without focal necrosis). Notably, in contrast to granular neurons, there is not a consistent deficit in unipolar, bipolar, multipolar, or inverted pyramidal neurons in the white matter or subplate region in PVL. The finding of reduced density of white matter neurons in the necrotic foci in PVL is not unexpected, since necrosis involves destruction of all cellular elements. On the other hand, the deficit in the granular neurons distant from the focally necrotic lesions (i.e., in the subplate region), presumably in areas with less severe insult, is of major interest. The preferential damage to granular neurons, including those distant from the necrotic foci, suggests that this particular subtype is exquisitely sensitive to hypoxia-ischemia. Because of the critical roles of subplate neurons in cerebral cortical development, such injury could have important secondary deleterious effects in cortex (see later).
Radial Glial Fibers
Development of Radial Glial Fibers and Astrocytes in the Cerebral White Matter in the Preterm Period.
The last half of human gestation is a crucial time in astrocyte formation in the cerebral cortex and white matter. The radial glial cell originates in the ventricular zone/subventricular zone (SVZ), retains connections with the ependyma and pia, and is capable of generating neurons and astrocytes ( Table 14.2 ). Early in development these cells are important neuronal progenitors (see Chapter 5 ). Radial glial cells have long, thin, linear processes (i.e., radial glial fibers [RGFs]), which serve as a guide for migrating neuroblasts and glial cells. Glutamatergic neurons form in the dorsal telencephalic pallium and migrate along RGFs early in gestation (see Chapter 6 ). In the human brain, in contrast to the rodent brain, approximately two thirds of GABAergic neurons arise from the dorsal telencephalic zone and migrate along RGFs; the remaining one third originate in the GE and migrate tangentially to the cortex (see Chapter 5 ). From 19 to 30 weeks, RGFs are abundant; around 30 to 31 weeks, they begin to transform into fibrous astrocytes in the white matter; and from 30 weeks to term, they progressively disappear as the white matter becomes increasingly populated with transformed astrocytes (see Table 14.2 ). By term, RGFs completely disappear, thereby definitively marking the end of radial migration. Fibrous astrocytes in the white matter also form from glial precursors that migrate outward from the ventricular zone/SVZ independent of RGFs. Reactive gliosis with gemistocytic morphology and immunostaining positive for glial fibrillary acidic protein (GFAP) begins around midgestation in the human brain. There is presumed loss and fragmentation of RGFs transected by focal periventricular necrosis (see Table 14.2 ). RGF damage could adversely affect radial neuronal migration with secondary maldevelopment of the vertical columns of the cerebral cortex. This idea of diffuse loss of RGFs has not been rigorously tested, however, in the preterm brain with the necessary tissue methods to define quantitative derangements in cortical mini- and macrocolumn formation. Damage to RGFs may also potentially impair astrocytic development, as fibrous astrocytes in the white matter develop from the transformation of RGFs, and protoplasmic astrocytes in the cortex transform from layer I astrocytes following RGF migration. A deficit in fibrous and/or protoplasmic astrocytes in the encephalopathy of prematurity may potentially be masked by gliosis, as there are no quantitative criteria for an adequate astrocytic response. Nevertheless, reactive astrocytes in the encephalopathy of prematurity demonstrate evidence of oxidative and nitrative stress, which is potentially primary and could lead to an inadequate glial response. Indeed, “acutely damaged glia” in PVL may represent astrocytes undergoing cell death. Given the role of astrocytes in protecting against ischemic injury via glutamate uptake and in orchestrating cytokine responses, damage to them secondary to potential RGF injury in the encephalopathy of prematurity could be especially deleterious. The delineation of RGF pathology in the encephalopathy of prematurity is an important direction for future research.
|
White Matter Axons
Diffuse Axonal Injury in the Cerebral White Matter in the Encephalopathy of Prematurity.
With β-amyloid precursor protein, axonal spheroids are detected within the necrotic lesions of PVL, whether focal or large ( Fig. 14.9 ). This finding is not unexpected in view of the injury to all cellular elements in the focal areas of necrosis. However, surprisingly perhaps, in the more diffuse nonnecrotic component of PVL, evidence for axonal injury has recently been gathered . With the apoptotic marker fractin, diffuse axonal injury is detected in the white matter distant from acute or organizing necrotic foci, suggesting a widespread axonopathy in PVL ( Fig. 14.10 ). This diffuse axonal damage could reflect secondary degeneration of thalamocortical afferents complicating primary thalamic neuronal loss. Alternatively, hypoxic-ischemic or inflammatory injury directly to the axon, with secondary impairments in axonal-OL interactions in the initiation and maintenance of myelination, seems possible (see later). Irrespective of its pathogenesis, widespread axonal damage likely contributes to the reduced white matter volume and callosal thinning in end-stage PVL. Axonal injury throughout the diffuse and focal components of PVL may also lead to architectonic changes in the overlying cerebral cortex.
