Encephalopathy of Prematurity: Neuropathology

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. 18.2 ).

The necrotic foci likely represent a core infarct with destruction of all cellular elements , whereas 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. 18.3 ). 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 on the data set, have only microscopic necrotic foci (with macrophagocytic infiltration) that measure less than 2 mm in diameter ( Fig. 18.4 ). 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. 18.5 ). 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 20 ), although careful anatomical imaging correlations are lacking.

Diffuse white matter gliosis (DWMG) without periventricular necrotic foci occurs frequently in preterm brains, but its relationship to PVL has not been fully clarified, although the bulk of current data supports the notion that it represents the least severe end of a spectrum of ischemic injury to the premyelinated white matter, with PVL at the most severe end ( Fig. 18.6 ). Reactive gliosis and activated microglia are the two major inflammatory components of PVL ( Fig. 18.7 ). 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 to the intragyral white matter and thereby suggests a role for injury in the vascular distal fields of the cerebral white matter. Activated microglia likewise conform to this regional distribution, whereas 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, for example, reactive astrocytes in PVL express interferon-γ and thus are a potential source for this cytokine, which is toxic particularly to pre-OLs compared with mature OLs.

Reactive astrocytes and microglia/macrophages also help 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 compared with control white matter; moreover, macrophages in the necrotic foci express EAAT2. Yet reactive astrocytes and microglia also may contribute to free radical injury in PVL, as indicated by intense expression of inducible nitric oxide synthase (iNOS), a marker of nitrative stress, both in reactive astrocytes in the acute through chronic stages of PVL, and in activated microglia primarily in the acute stage. The latter observation suggests 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. 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. 18.8 ). In addition, F(2)-isoprostanes, an arachidonate metabolite/lipid peroxidation marker of oxidative damage, are significantly increased in the white matter of early PVL cases.

The cellular evolution of PVL involves acute loss of pre-OLs ; some OL cell bodies appear to survive but with loss of cell processes. 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 show an attempt at replenishment of pre-OLs by proliferation of progenitors by an unknown stimulus, but these newly generated pre-OLs fail to mature. The cellular result is dominance of pre-OLs over mature OLs (see later and Chapter 19 ). 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 abnormalities 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. The impaired maturation of the newly generated pre-OLs may be due to the presence of activated microglia and reactive astrocytes that make up the diffuse component of PVL as these cells may have deleterious effects on dysmaturational events pertaining to pre-OLs, neurons, and axons.

These dysmaturational events occur after injury, in part, because microglia have important roles in normal brain development and are critical to key aspects of OL differentiation, synaptogenesis, synaptic pruning, axonal development, and myelination. When activated as part of a PVL-associated neuroinflammatory response, microglia release the aforementioned proinflammatory cytokines and free radicals and promote excitotoxicity, all of which are damaging, particularly to pre-OLs and axons, and can derail the normal developmental functions of microglia, leading to perturbations in synaptogenesis, axonal guidance, and neural circuit formation.

Astrocytes, the other key cell type of the diffuse component in PVL, are critical in a number of important developmental processes, some of which are similar to or overlap with microglial functions. During the last half of gestation fibrous astrocytes, derived from radial glia, increase in cerebral white matter. Astrocytes are critical to axonal guidance and pruning, the blood-brain barrier, neuronal survival, angiogenesis, synaptogenesis, and synaptic pruning. Astrocytes respond to hypoxia-ischemia and inflammation by developing a “reactive” phenotype that is capable of damaging other cells, including pre-OLs, thereby contributing to dysmaturational events. Proinflammatory activated microglia have been shown to induce reactive astrocytes that can injure brain tissue.

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. 18.9 ). 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 19 ). 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%). 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). Perhaps more likely, the dysmaturational abnormalities described in the next sections are not readily detectable by conventional neuropathological techniques.

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. 18.10 ). 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 ) ( Fig. 18.11 ). 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. However, in a study of PVL cases compared with 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). Rather than excitotoxicity, 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 γ-aminobutyric acid (GABA)ergic interneurons that play a key role in cortical specification, output, and synaptic plasticity (see Chapter 7 ) ( Fig. 18.12 ). 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. Neuropathologic studies of infants with moderate to severe WMI reported decreases in central white matter neuron populations, a finding that would be consistent with reduction in late migrating GABAergic neurons. Reduced cortical surface area and volume, as well as reductions in cortical gyration and perturbations in neural connectivity, would be expected to result from the loss of these neurons. These abnormalities have been well documented subsequent to premature birth and cerebral WMI (see Chapter 20 ).