The topography of the lesions was uniform, primarily affecting the white matter in a zone within the subcallosal, superior fronto-occipital and superior longitudinal fasciculi, the external and internal border zone of the temporal and occipital horn of the lateral ventricles, and some parts of the corona radiate.⁵¹ These areas appeared pale, usually bilateral, but without definite symmetry (Figure 59-1). Although not unanimously accepted, it has been noted that the anatomic distribution of PVL correlates with the development of perforating medullary arteries and areas that represent arterial border or end zones, that arise between ventriculopetal and ventriculofugal arteries within the deep white matter (Figure 59-2).⁷¹ Immunohistochemical studies further confirm a low vessel density in the deep white matter between 28 to 36 weeks’ gestation, whereas in the subcortical white matter, the vessel density is low between 16 and 24 weeks and thereafter increases (Figure 59-3).¹¹⁴

New Neuropathologic Insights

This diffuse white matter damage is macroscopically characterized by a paucity of white matter, thinning of the corpus callosum, and in later stages, ventriculomegaly and delayed myelination.53,89 With the use of immunocytochemical techniques, the assessment of autopsy tissue has allowed further localization of cell-specific injury in white matter damage. Deep periventricular white matter is prone to show focal necrosis regionally consistent with the presumed vascular end zones/border zones, whereas in the peripheral white matter, diffuse injury could be characterized by preferential death or injury of late oligodendrocyte progenitors and immature oligodendrocytes or pre-OLs (see Chapter 58).¹⁰ Recent postmortem data support the hypothesis that in very preterm infants, blockade of maturation of oligodendrocytes, rather than their death, is the key neuropathologic hallmark in diffuse white matter damage.^17,148 In addition, more recent neuropathologic studies on human preterm material have shown the extensive involvement of axonal damage,⁶⁰ especially thalamocortical fibers and damage to white matter neuronal populations (GABAergic interneurons),⁸⁴ and damage to the cerebellar white matter.⁸³

Vulnerability of Oligodendroglia Cell Line.

Several lines of evidence implicate damage to immature oligodendrocytes during a specific window of vulnerability as a significant underlying factor in the pathogenesis of PVL (see Models of Encephalopathy of Prematurity). Oligodendrocyte progenitor cells proliferate and die by programmed cell death (see Chapter 58) regulated by trophic factors such as platelet-derived growth factor and insulin-like growth factor.¹² The activation of cytokine receptors on the surface of oligodendrocytes can lead to the death or maturation blockade of these cells. Studies in vitro have shown that the inflammatory cytokines TNF-α and interferon-gamma are toxic to cultured oligodendrocyte progenitor cells.⁵ Selective injury to oligodendrocytes is mediated by induction of “death” receptors such as Fas on the surface of oligodendrocytes. Direct axonal contact appears to be another important factor for the survival and maturation of oligodendrocytes.²² Oligodendrocytes are further susceptible to oxidative damage mediated by free radicals such as reactive oxygen and nitrogen species and as a consequence of the depletion of the main antioxidant, glutathione.⁸ Injury-induced swelling and disruption to axons within the white matter leads to locally elevated glutamate, which also induces oligodendrocyte cell death and/or injury. Glutamate toxicity depends on the maturational stage of the oligodendrocyte and is mediated via the alpha-3-amino-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor and potentially through N-methyl-d-aspartate (NMDA) receptors.^34,79

Experimental data show, on the other hand, important increase in NG2 (high molecular weight, integral membrane chondroitin sulphate proteoglycan)-positive oligodendrocyte progenitor cells within the area of the injury.¹³⁶ The role of this increase in NG2 cells is currently unknown, but this population is distinct from neurons, oligodendrocytes, astrocytes, and microglia. This cell population could comprise multipotent cells capable of differentiating in any other type of cells and playing a role in axonal growth and myelination, and in regeneration after injury with functional integration in neural circuitry.

Microglial Activity.

Specific immunocytochemical markers (e.g., CD68) have identified a marked increase of activated microglia in diffuse white matter injury.⁶¹ Microglia are already widely dispersed throughout the immature white matter by 22 weeks’ gestation. These cells are fully capable of producing potentially toxic inflammatory mediators, free radicals, and reactive oxygen intermediates.¹²⁹ The phagocytic activity of microglia and their capacity for oxidative mediated injury are potently enhanced by inflammatory mediators (IFN-γ, TNF-α, IL-β and bacterial lipopolysaccharide [LPS]).¹³⁹ Studies of preoligodendrocytes of the same maturational stage as those populated in the immature white matter of the human premature infant show that cells are exquisitely vulnerable to attack by reactive oxygen species and reactive nitrogen species produced by activated microglia.¹¹⁰ Presence of activated microglia inducing cell death in immature white matter, both in preoligodendrocytes as well as in astrocytes, has been widely confirmed.^39,140 So far it also seems that microglia and resident mononuclear phagocytes are the primary sources for the proinflammatory cytokines in PVL brains.⁸⁰

Neuronal/Axonal Damage.

Indirect assessment of axonal damage in classical PVL was done by immunostaining for beta amyloid precursor protein, a neuronoaxonal protein. Immunostaining of damaged axons was predominant in the acute phase of PVL and was no longer detectable in the chronic stage.¹⁰⁸ Swollen axons calcify (probably owing to glutamatergic overactivation), accumulate iron, and degenerate; this has been shown to occur without overt coagulation necrosis of all tissue components.⁶² Axons from corticospinal tract, thalamocortical fibers, optic radiation, superior occipitofrontal fasciculus, and the superior longitudinal fasciculus may be affected and result in motor, sensory, visual, and higher cortical functions deficits. Thalamocortical projections that course through the white matter develop prior to the functional development of cortical neurons. Therefore, the ensuing disruption to these circuits and to the subcortical plate may not only affect the function, but also the density, survival, and organization of cortical neurons and the cortex itself^70,86 (see Subplate Damage).

In addition, there is evidence for cell death of progenitor cells, not neural stem cells in the subventricular zone after hypoxia-ischemia.⁴⁷ These destructive effects are paralleled with potential trophic reactions such as the proliferation of potentially pluripotent progenitors cells, also called polydendrocytes in the area of injury.¹³⁵ These cells are known to differentiate into oligodendrocytes and, to a lesser extent, into gray matter astrocytes and potentially even neurons.¹¹⁸

Recent studies also suggest that preterm delivery decreases the number of interneurons in some cortical areas, suggesting that their production, migration, and/or survival is impaired.¹⁴⁹ Activated microglia in the white matter could contribute to impairment of migration and/or survival of interneurons.⁹¹

Subplate Damage.

Damage to the early developing subplate neurons, with their critical role for the organization of the cortical plate (see Chapter 58), has long been postulated as a possible mechanism by which injury to the immature brain results in long-lasting motor and cognitive deficits.¹⁵² McQuillen et al.¹⁰⁷ were able to show specific cell death in subplate neurons after hypoxia-ischemia in very immature animals. Lack of guidance for the thalamocortical connections ensued by the loss of subplate neurons may represent one of the major developmental disturbances after injury to the immature brain.⁸⁷

The current concept of pathogenesis of encephalopathy of prematurity is based on the combination of destructive and developmental disturbances, which are schematically summarized in Figure 59-4.

Hypoperfusion and Hypoxia-Ischemia

Hypoxic-ischemic or hypoperfusion insults have been used in a large variety of neonatal species including rats, mice, rabbits, pigs, and dogs. In most cases, these insults produce specific gray matter damage (mimicking lesions observed in human full-term infants), which has been the focus of most of these studies, whereas observed white matter damage is generally considered as an extension of gray matter damage in the most severe cases. However, notable exceptions highlight some potentially important features of perinatal white matter damage.

In dog pups, hypoxic-ischemic insult by bilateral carotid ligation selectively induces white matter damage mimicking human PVL.¹⁶⁰ This selective vulnerability of white matter could underlie a genetic predisposition of the dog to white matter hypoxic-ischemic damage. Modern tools of genomics could unravel important genes involved in the pathophysiology of perinatal white matter damage.

In rats, the classical Rice-Vannucci model performed on postnatal day 7 or 9 predominantly leads to gray matter lesions. However, analyses have shown the involvement of the periventricular white matter with the involvement of immature oligodendroglial and progenitor cells.9,48,137 More important, adaptation of this paradigm to more immature newborn rats (postnatal days 1 or 3) has allowed production of important lesions in the periventricular white matter with relative sparing of the classical intracortical injuries (Figure 59-6).^21,107,136 These studies have also highlighted the specific involvement of subplate neurons in brain damage. Altogether, these data obtained in newborn rats further support the notion of an ontogenic window of white matter sensitivity and subplate vulnerability to insults such as hypoxia-ischemia.

Asphyxia of sheep fetuses (around 65% of gestation) has been shown to induce periventricular (focal and diffuse) and subcortical (diffuse) white matter disease, accompanied by acute astrocyte and oligodendrocyte loss, and marked reactive microgliosis.¹⁰¹ These studies support the concept of the association of focal (such as cystic lesions) and diffuse (such as diffuse microglial activation) white matter lesions, the pathophysiology of which is potentially distinct. Furthermore, it was shown by microdialysis that white matter glutamate levels were significantly increased following asphyxia, supporting a role of excitotoxicity in the pathogenesis of such white matter lesions (see Excitotoxicity).⁹⁷

Exposure of pregnant rats¹⁵ or postnatal rat pups¹⁴⁵ to hypoxia induces pathologic changes in the periventricular white matter that are reminiscent of human periventricular leukomalacia, with inflammation, astrogliosis, and myelination delay in the prenatal model, and white matter atrophy, ventriculomegaly, and alteration of synaptic maturation in the postnatal paradigm. Although the initial insult is a pure hypoxia, the observed effects are likely the result of the combination of different mechanisms such as hypoperfusion, ischemia, inflammation, and/or oxidative stress induced by the protracted hypoxia and the subsequent reoxygenation phase.

Infection and Inflammation

Systemic administration of LPS to immature cats, dogs, or rabbits induces white matter lesions.4,50 Systemic administration of LPS can induce a marked systemic inflammation and immune changes in the central nervous system, such as an increased expression of CD14. Furthermore, high doses of LPS can induce hypotension, hypoglycemia, hyperthermia, and lactic acidosis, all potential factors predisposing to brain damage. However, low doses of LPS, which do not induce significant hypotension, were also shown to induce white matter damage in fetal sheep.^58,100 In fetal sheep, the comparison of white damage induced by cord occlusion and by LPS injection revealed distinct patterns of microglia-macrophage activation, suggesting separate or partly separate underlying mechanisms.¹⁰⁰ On the other hand, systemic administration of LPS to newborn rats failed to induce detectable white matter lesions, although increased cytokine production and microglial activation were observed in white matter.^20,45,158 These data suggest species differences that might be linked to species-related genetic susceptibility of the developing white matter to infectious-inflammatory factors. Interestingly, in vitro studies have shown that macrophages are necessary for LPS to induce preoligodendrocyte cell death.

Live infectious agents have also been used by a few research groups to produce models of white matter lesions. In pregnant rabbits, ascending intrauterine infection with Escherichia coli (E. coli) caused focal white matter damage in 6% of live fetuses,¹⁵⁹ whereas direct inoculation of E. coli in the uterine cavity combined with early antibiotics produced focal white matter cysts in about 20% of live fetuses and diffuse white matter cell death in almost all live fetuses (Figure 59-7).³¹ Cystic lesions are accompanied by macrophage-microglia activation and reactive astrogliosis, whereas diffuse white matter cell death does not induce such glial responses. These results suggest that these two types of brain damage have distinct pathophysiologic mechanisms.

In addition, a study has shown that infection of pregnant mice with Ureaplasma parvum, a germ rather frequently isolated in chorioamnionitis, induces central microgliosis and disrupted brain development as detected by decreased number of calbindin-positive and calretinin-positive neurons in the neocortex, as well as myelination defect in the periventricular white matter.¹¹⁹

Intrauterine inoculation of Border disease virus to pregnant sheep induces decreased expression of white matter molecules, including myelin basic protein in the fetuses.³ However, the virus also infects the thyroid and the pituitary gland, raising the question of the precise etiology of the white matter damage (low thyroid hormones versus infectious-inflammatory insult).

Finally, a recent study showed that exposure of newborn mice to low doses of systemic IL-1β, induces a moderate and transient inflammatory response during the neonatal period that is sufficient to disrupt oligodendrocyte maturation, myelin formation, and axonal development.⁴⁶ These white matter abnormalities are moderate during the developmental period but do persist until adulthood. They lead to permanent deficiencies in cognitive tests. The underlying molecular mechanisms include blockade of microglial activation, oligodendrocyte maturation blockade, and alterations of the transcription of genes implicated in oligodendrogenesis, myelin formation, and axonal maturation.

Excitotoxicity and Oxidative Stress

Glutamate can act on several types of receptors, including NMDA, AMPA, kainate, and metabotropic receptors. Excess release of glutamate has been suggested to represent a molecular mechanism common to some of the risk factors for brain lesions associated with cerebral palsy. In keeping with this possibility, injection of glutamate agonists into the striatum, neocortex, or periventricular white matter of newborn rodents (rats, mice, or hamsters), rabbits, or kittens produces, according to the stage of brain maturation, histologic lesions that mimic those seen in humans with cerebral palsy, such as neuronal migration disorders, polymicrogyria, cystic periventricular leukomalacia, and hypoxic-ischemic or ischemic-like cortical and striatal lesions.1,49,78,103,106

Studies exploring the pathophysiology of these excitotoxic white matter lesions in newborn rodents and rabbits have permitted the following contributions (Figure 59-8) ^{40,49,104,128,141}:

Extensive in vitro studies have confirmed the exquisite susceptibility of preoligodendrocytes to AMPA-kainate agonists and to oxidative stress.⁷⁹

Combined Insults

To further support the hypothesis of a multifactorial hypothesis of perinatal brain damage (see Figure 59-5), different groups have combined insults in newborn rodents.

Pretreatment of newborn mice with systemic proinflammatory cytokines (e.g. IL-1-beta, IL-6 or TNF-alpha) before an excitotoxic insult significantly exacerbates excitotoxic white matter lesions, demonstrating a causative link between circulating proinflammatory cytokines and white matter damage.³⁸ Results suggest that this effect of proinflammatory cytokines is more pronounced with NMDA receptor agonists when compared with AMPA-kainate receptor agonists (see Figure 59-8). The precise mechanism by which these cytokines systemically act on white matter excitotoxicity remains to be determined, but could potentially involve activation of brain cyclooxygenase or activation of microglia with increased white matter production of reactive oxygen species and cytokines.

Similarly, systemic pretreatment with IL-9, a Th2 cytokine, was shown to exacerbate NMDA receptor-mediated white matter lesions38,123 The mechanism of IL-9 toxicity involves brain mast cell degranulation and excess release of histamine. Interestingly, increased circulating levels of IL-9 around birth had been demonstrated in a subgroup of human infants who later developed cerebral palsy.¹¹⁷ Chronic mild stress of pregnant mice has been shown to induce a significant exacerbation of excitotoxic white matter lesions in pups. LPS was also used to sensitize the newborn brain to hypoxia-ischemia. A low dose of this endotoxin, given 4 hours before a mild hypoxic-ischemic insult in postnatal day 7 (P7) rats induced extensive brain damage, whereas each insult given separately did not induce any detectable brain lesion.⁴⁵

Recent years have seen the emergence of several experimental paradigms to study white matter diseases of the preterm infant. These animal models have permitted identification of some of the potentially key cellular (e.g., preoligodendrocytes, microglia-macrophages) and molecular (glutamate, cytokines, and other inflammatory mediators, reactive oxygen species, etc.) players involved in the pathophysiology of perinatal brain damage. These studies have also delineated potential targets for neuroprotection. Future studies should incorporate imaging and behavioral studies to facilitate comparisons with the human situation. Although it is clear that studies with rodents, owing to their cost and availability, are absolutely necessary to generate and test multiple hypotheses, the use of larger models such as sheep, pigs, and monkeys will remain a key for testing in a more reliable fashion the relevance of the obtained data for the human situation.