Brain injury in premature infants is of enormous public health importance because of the large number of such infants who survive with serious neurodevelopmental disability, including major cognitive deficits and motor disability. This type of brain injury is generally thought to consist primarily of periventricular leukomalacia (PVL), a distinctive form of cerebral white matter injury. Important new work shows that PVL is frequently accompanied by neuronal/axonal disease, affecting the cerebral white matter, thalamus, basal ganglia, cerebral cortex, brain stem, and cerebellum. This constellation of PVL and neuronal/axonal disease is sufficiently distinctive to be termed “encephalopathy of prematurity”. The thesis of this Review is that the encephalopathy of prematurity is a complex amalgam of primary destructive disease and secondary maturational and trophic disturbances. This Review integrates the fascinating confluence of new insights into both brain injury and brain development during the human premature period.

Innate immunity is an evolutionarily ancient system that provides organisms with immediately available defense mechanisms through recognition of pathogen-associated molecular patterns. We show that in the CNS, specific activation of innate immunity through a Toll-like receptor 4 (TLR4)-dependent pathway leads to neurodegeneration. We identify microglia as the major lipopolysaccharide (LPS)-responsive cell in the CNS. TLR4 activation leads to extensive neuronal death in vitro that depends on the presence of microglia. LPS leads to dramatic neuronal loss in cultures prepared from wild-type mice but does not induce neuronal injury in CNS cultures derived from tlr4 mutant mice. In an in vivo model of neurodegeneration, stimulating the innate immune response with LPS converts a subthreshold hypoxic-ischemic insult from no discernable neuronal injury to severe axonal and neuronal loss. In contrast, animals bearing a loss-of-function mutation in the tlr4 gene are resistant to neuronal injury in the same model. The present study demonstrates a mechanistic link among innate immunity, TLRs, and neurodegeneration. Systemic infection is associated with sustained worsening in many diseases of the CNS, yet the molecular and cellular relationship between infection outside the CNS and potential neuronal loss within the CNS is elusive. Activation of microglia, bone marrow-derived macrophage-like cells that function as the resident immune defense system of the brain (1), is a characteristic feature of most neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, multiple sclerosis, AIDS dementia complex, and amyotrophic lateral sclerosis as well as ischemia and posttraumatic brain injury (2-4). Neurotoxicity induced by ␤-amyloid or HIV proteins in mixed CNS cultures depends on the presence and activation of microglia (5, 6). Liberatore et al. (7) demonstrated in vivo that microglial inducible nitric oxide synthase plays a crucial role in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration in the MPTP mouse model of Parkinson's disease.The evolutionarily ancient innate immune system provides the first line of host defense against a large variety of pathogens and also controls many aspects of the adaptive immune response (8). Cells of the innate immune system recognize invariant molecular structures of pathogens termed pathogen-associated molecular patterns through a series of genetically conserved and stable cell-surface receptors related to the Drosophila gene toll that thus are referred to as Toll-like receptors (TLRs) (9).TLR4 functions as the signal-transducing receptor for the endotoxin lipopolysaccharide (LPS) (10), which is a major component of the outer membrane of Gram-negative bacteria. LPS binds to the serum protein LPS-binding protein and the soluble or glycosylphosphatidylinositol-anchored CD14. This complex in turn binds to TLR4 (11) and initiates an intracellular signaling pathway that regulates gene expression through derepression of the transcriptional a...

Hypoxic-ischemic injury to the periventricular cerebral white matter [periventricular leukomalacia (PVL)] results in cerebral palsy and is the leading cause of brain injury in premature infants. The principal feature of PVL is a chronic disturbance of myelination and suggests that oligodendrocyte (OL) lineage progression is disrupted by ischemic injury. We determined the OL lineage stages at risk for injury during the developmental window of vulnerability for PVL (23-32 weeks, postconceptional age). In 26 normal control autopsy human brains, OL lineage progression was defined in parietal white matter, a region of predilection for PVL. Three successive OL stages, the late OL progenitor, the immature OL, and the mature OL, were characterized between 18 and 41 weeks with anti-NG2 proteoglycan, O4, O1, and anti-myelin basic protein (anti-MBP) antibodies. NG2ϩO4ϩ late OL progenitors were the predominant stage throughout the latter half of gestation. Between 18 and 27 weeks, O4ϩO1ϩ immature OLs were a minor population (9.9 Ϯ 2.1% of total OLs; n ϭ 9). Between 28 and 41 weeks, an increase in immature OLs to 30.9 Ϯ 2.1% of total OLs (n ϭ 9) was accompanied by a progressive increase in MBPϩ myelin sheaths that were restricted to the periventricular white matter. The developmental window of high risk for PVL thus precedes the onset of myelination and identifies the late OL progenitor as the major potential target. Moreover, the decline in incidence of PVL at ϳ32 weeks coincides with the onset of myelination in the periventricular white matter and suggests that the risk for PVL is related to the presence of late OL progenitors in the periventricular white matter.

Brain injury in the premature infant is a problem of enormous importance. Periventricular leukomalacia (PVL) is the major neuropathologic form of this brain injury and underlies most of the neurologic morbidity encountered in survivors of premature birth. Prevention of PVL now seems ultimately achievable because of recent neurobiologic insights into pathogenesis. The pathogenesis of this lesion relates to three major interacting factors. The first two of these, an incomplete state of development of the vascular supply to the cerebral white matter, and a maturation-dependent impairment in regulation of cerebral blood flow underlie a propensity for ischemic injury to cerebral white matter. The third major pathogenetic factor is the maturation-dependent vulnerability of the oligodendroglial (OL) precursor cell that represents the major cellular target in PVL. Recent neurobiologic studies show that these cells are exquisitely vulnerable to attack by free radicals, known to be generated in abundance with ischemia-reperfusion. This vulnerability of OLs is maturation-dependent, with the OL precursor cell highly vulnerable and the mature OL resistant, and appears to relate to a developmental window characterized by a combination of deficient antioxidant defenses and active acquisition of iron during OL differentiation. The result is generation of deadly reactive oxygen species and apoptotic OL death. Important contributory factors in pathogenesis interact with this central theme of vulnerability to free radical attack. Thus, the increased likelihood of PVL in the presence of intraventricular hemorrhage could relate to increases in local iron concentrations derived from the hemorrhage. The important contributory role of maternal/fetal infection or inflammation and cytokines in the pathogenesis of PVL could be related to effects on the cerebral vasculature and cerebral hemodynamics, to generation of reactive oxygen species, or to direct toxic effects on vulnerable OL precursors. A key role for elevations in extracellular glutamate, caused by ischemia-reperfusion, is suggested by demonstrations that glutamate causes toxicity to OL precursors by both nonreceptor-and receptor-mediated mechanisms. The former involves an exacerbation of the impairment in antioxidant defenses, and the latter, an ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor-mediated cell death. Most importantly, these new insights into the pathogenesis of PVL suggest potential preventive interventions. These include avoidance of cerebral ischemia by detection of infants with impaired cerebrovascular autoregulation, e.g. through the use of in vivo near-infrared spectroscopy, the use of free radical scavengers to prevent toxicity by reactive oxygen species, the administration of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor antagonists to prevent glutamatemediated injury, or the use of maternal antibiotics or anticytokine agents to prevent toxicity from maternal/fetal infection or inflammation and cytokines.

This MRI study of prematurely born infants further defines the nature of quantitative cerebral structural abnormalities present as early as term equivalent. The abnormalities particularly involve cerebral neuronal regions including both cortex and deep nuclear structures. The pattern of cerebral alterations is related most significantly to the degree of immaturity at birth and to concomitant WM injury. The alterations are followed by abnormal short-term neurodevelopmental outcome.

Alterations of the architecture of cerebral white matter in the developing human brain can affect cortical development and result in functional disabilities. A line scan diffusion-weighted magnetic resonance imaging (MRI) sequence with diffusion tensor analysis was applied to measure the apparent diffusion coefficient, to calculate relative anisotropy, and to delineate three-dimensional fiber architecture in cerebral white matter in preterm (n = 17) and full-term infants (n = 7). To assess effects of prematurity on cerebral white matter development, early gestation preterm infants (n = 10) were studied a second time at term. In the central white matter the mean apparent diffusion coefficient at 28 wk was high, 1.8 microm2/ms, and decreased toward term to 1.2 microm2/ms. In the posterior limb of the internal capsule, the mean apparent diffusion coefficients at both times were similar (1.2 versus 1.1 microm2/ms). Relative anisotropy was higher the closer birth was to term with greater absolute values in the internal capsule than in the central white matter. Preterm infants at term showed higher mean diffusion coefficients in the central white matter (1.4 +/- 0.24 versus 1.15 +/- 0.09 microm2/ms, p = 0.016) and lower relative anisotropy in both areas compared with full-term infants (white matter, 10.9 +/- 0.6 versus 22.9 +/- 3.0%, p = 0.001; internal capsule, 24.0 +/- 4.44 versus 33.1 +/- 0.6% p = 0.006). Nonmyelinated fibers in the corpus callosum were visible by diffusion tensor MRI as early as 28 wk; full-term and preterm infants at term showed marked differences in white matter fiber organization. The data indicate that quantitative assessment of water diffusion by diffusion tensor MRI provides insight into microstructural development in cerebral white matter in living infants.

The immediate or innate immune response is the first line of defense against diverse microbial pathogens and requires the expression of recently discovered toll-like receptors (TLRs). TLR4 serves as a specific receptor for lipopolysaccharide (LPS) and is localized on the surface of a subset of mammalian cells. Although innate immunity is a necessary host defense against microbial pathogens, the consequences of its activation in the CNS can be deleterious, as we show here in a developing neural model. We examined the major non-neuronal cell types in the CNS for expression of TLR4 and found that microglia expressed high levels, whereas astrocytes and oligodendrocytes expressed none. Consistent with TLR4 expression solely in microglia, we show that microglia are the only CNS glial cells that bind fluorescently tagged lipopolysaccharide. Lipopolysaccharide led to extensive oligodendrocyte death in culture only under conditions in which microglia were present. To determine whether TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte death in mixed glial cultures, we studied cultures generated from mice bearing a loss-of-function mutation in the tlr4 gene. Lipopolysaccharide failed to induce oligodendrocyte death in such cultures, in contrast to the death induced in cultures from wild-type mice. Finally, stereotactic intracerebral injection of lipopolysaccharide into the developing pericallosal white matter of immature rodents resulted in loss of oligodendrocytes and hypomyelination and periventricular cysts. Our data provide a general mechanistic link between (1) lipopolysaccharide and similar microbial molecular motifs and (2) injury to oligodendrocytes and myelin as occurs in periventricular leukomalacia and multiple sclerosis.

Cerebral white matter injury, characterised by loss of premyelinating oligodendrocytes (pre-OLs), is the most common form of injury to the preterm brain and is associated with a high risk of neurodevelopmental impairment. The unique cerebrovascular anatomy and physiology of the premature baby underlies the exquisite sensitivity of white matter to the abnormal milieu of preterm extrauterine life, in particular ischaemia and inflammation. These two upstream mechanisms can coexist and amplify their effects, leading to activation of two principal downstream mechanisms: excitotoxicity and free radical attack. Upstream mechanisms trigger generation of reactive oxygen and nitrogen species. The pre-OL is intrinsically vulnerable to free radical attack due to immaturity of antioxidant enzyme systems and iron accumulation. Ischaemia and inflammation trigger glutamate receptor-mediated injury leading to maturation-dependent cell death and loss of cellular processes. This review looks at recent evidence for pathogenetic mechanisms in white matter injury with emphasis on targets for prevention and treatment of injury.Cerebral white matter injury in the premature infant is a problem of enormous importance. For example, in the USA each year approximately 60 000 infants (1.5% of the 4 000 000 yearly live births) are born with a birth weight less than 1500 g, 1 and based on MRI data at least 50% exhibit some degree of cerebral white matter injury, 2, 3 as defined later. This injury likely accounts for the predominance of neurological deficits observed in the approximately 90% of infants who survive. These deficits in survivors include cerebral palsy in 5-10% and importantly, cognitive/behavioural/attentional deficits in about 50%. 4, 5 Although other pathologies occur in premature infants-for example, severe intraventricular haemorrhage, periventricular haemorrhagic infarction, hydrocephalus, cerebellar disease-cerebral white matter injury seems to be the predominant lesion. Prevention of this injury requires insight into pathogenesis, and recent research holds promise that preventive interventions will be found. PERIVENTRICULAR LEUKOMALACIA AND ENCEPHALOPATHY OF PREMATURITYCerebral white matter injury is the term used in this review for the full spectrum of periventricular leukomalacia (PVL). PVL has two components-that is, focal necrosis deep in the white matter with loss of all cellular elements, and a more diffuse component in central cerebral white matter with loss of pre-myelinating oligodendrocytes (pre-OLs), astrogliosis and microglial infiltration. 2 PVL occurs in two overlapping forms: cystic PVL, in which the focal necroses are macroscopic and evolve to multiple cysts ( fig 1A); and non-cystic PVL, in which the focal necroses are microscopic and evolve principally to glial scars ( fig 1B). A third form of cerebral white matter abnormality consists of diffuse astrogliosis without focal necroses ( fig 1C). That the latter is the mildest form of injury in a spectrum that includes cystic Correspondence to: Dr J J ...