Robin L. Haynes

Myelin Abnormalities without Oligodendrocyte Loss in Periventricular Leukomalacia

The cellular basis of myelin deficits detected by neuroimaging in long‐term survivors of periventricular leukomalacia (PVL) is poorly understood. We tested the hypothesis that oligodendrocyte lineage (OL) cell density is reduced in PVL, thereby contributing to subsequent myelin deficits. Using computer‐based methods, we determined OL cell density in sections from 18 PVL and 18 age‐adjusted control cases, immunostained with the OL‐lineage marker Olig2. Myelination was assessed with myelin basic protein (MBP) immunostaining. We found no significant difference between PVL and control cases in Olig2 cell density in the periventricular or intragyral white matter. We did find, however, a significant increase in Olig2 cell density at the necrotic foci, compared with distant areas. Although no significant difference was found in the degree of MBP immunostaining, we observed qualitative abnormalities of MBP immunostaining in both the diffuse and necrotic components of PVL. Abnormal MBP immunostaining in PVL despite preserved Olig2 cell density may be secondary to arrested OL maturation, damage to OL processes, and/or impaired axonal‐OL signaling. OL migration toward the “core” of injury may occur to replenish OL cell number. This study provides new insight into the cellular basis of the myelin deficits observed in survivors of PVL.

Axonal development in the cerebral white matter of the human fetus and infant

After completion of neuronal migration to form the cerebral cortex, axons undergo rapid elongation to their intra‐ and subcortical targets, from midgestation through infancy. We define axonal development in the human parietal white matter in this critical period. Immunocytochemistry and Western blot analysis were performed on 46 normative cases from 20–183 postconceptional (PC) weeks. Anti‐SMI 312, a pan‐marker of neurofilaments, stained axons as early as 23 weeks. Anti‐SMI 32, a marker for nonphosphorylated neurofilament high molecular weight (NFH), primarily stained neuronal cell bodies (cortical, subcortical, and Cajal‐Retzius). Anti‐SMI 31, which stains phosphorylated NFH, was used as a marker of axonal maturity, and showed relatively low levels of staining (approximately one‐fourth of adult levels) from 24–34 PC weeks. GAP‐43, a marker of axonal growth and elongation, showed high levels of expression in the white matter from 21–64 PC weeks and lower, adult‐like levels beyond 17 postnatal months. The onset of myelination, as seen by myelin basic protein expression, was ∼54 weeks, with progression to “adult‐like” staining by 72–92 PC weeks. This study provides major insight into axonal maturation during a critical period of growth, over an age range not previously examined and one coinciding with the peak period of periventricular leukomalacia (PVL), the major disorder underlying cerebral palsy in premature infants. These data suggest that immature axons are susceptible to damage in PVL and that the timing of axonal maturation must be considered toward establishing its pathology relative to the oligodendrocyte/myelin/axonal unit. J. Comp. Neurol. 484:156–167, 2005.

Oxidative and nitrative injury in periventricular leukomalacia: a review.

Periventricular leukomalacia (PVL) is the major substrate of cerebral palsy in survivors of prematurity. Its pathogenesis is complex and likely involves ischemia/reperfusion in the critically ill premature infant, with impaired regulation of cerebral blood flow, as well as inflammatory mechanisms associated with maternal and/or fetal infection. During the peak period of vulnerability for PVL, developing oligodendrocytes (OLs) predominate in the white matter. We hypothesize that free radical injury to the developing OLs underlies, in part, the pathogenesis of PVL and the hypomyelination seen in long‐term survivors. In human PVL, free radical injury is supported by evidence of oxidative and nitrative stress with markers to lipid peroxidation and nitrotyrosine, respectively. Evidence in normal human cerebral white matter suggests an underlying vulnerability of the premature infant to free radical injury resulting from a developmental mismatch of antioxidant enzymes (AOE) and subsequent imbalance in oxidant metabolism. In vitro studies using rodent OLs suggest that maturational susceptibility to reactive oxygen species is dependent, not only on levels of individual AOE, but also on specific interactions between these enzymes. Rodent in vitro data further suggest 2 mechanisms of nitric oxide damage: one involving the direct effect of nitric oxide on OL mitochondrial integrity and function, and the other involving an activation of microglia and subsequent release of reactive nitrogen species. The latter mechanism, while important in rodent studies, remains to be determined in the pathogenesis of human PVL. These observations together expand our knowledge of the role that free radical injury plays in the pathogenesis of PVL, and may contribute to the eventual development of therapeutic strategies to alleviate the burden of oxidative and nitrative injury in the premature infant at risk for PVL.

Development of microglia in the cerebral white matter of the human fetus and infant

Although microglial activation may be an initial beneficial response to a variety of insults, prolonged activation can release toxic substances and lead to cell death. Microglial activation secondary to hypoxia‐ischemia and/or infection in immature cerebral white matter is important in the pathogenesis of periventricular leukomalacia (PVL), the major pathological substrate of cerebral palsy in the premature infant. We hypothesize that a transient overexpression in activated microglial density occurs normally in the cerebral white matter of the human fetus during the peak window of vulnerability for PVL. Such an increase could render this region susceptible to insults that cause prolonged microglial activation, as conceptualized in PVL. To examine the developmental profile of microglia in the human fetus and infant brain, immunocytochemistry with microglial specific markers were used in 23 control (non‐PVL) cases ranging from 20 to 183 postconceptional (PC) weeks. Tomato lectin, used to identify microglial morphology, revealed that the cerebral white matter of the human fetus and infant is densely populated with intermediate and amoeboid microglia; the latter is indicative of an activated state. Quantitative analysis with CD68 showed increased density of activated microglia in the cerebral white matter of the fetus (<37 PC weeks) relative to the neonate/infant (≥37 PC weeks) and to the overlying cortex of either age group (P = 0.01). The primary finding of a transient, developmental‐dependent overabundance of CD68‐activated microglia in the cerebral white matter of the fetus suggests a potential “priming” of this area for diverse brain insults characterized by activation of microglia, particularly PVL. J. Comp. Neurol. 497:199–208, 2006.

Diffuse Axonal Injury in Periventricular Leukomalacia as Determined by Apoptotic Marker Fractin

Periventricular leukomalacia (PVL), the major substrate of neurologic deficits in premature infants, is associated with reduced white matter volume. Using immunomarkers of axonal pathology [β-amyloid precursor protein (β-APP) and apoptotic marker fractin], we tested the hypothesis that widespread (diffuse) axonal injury occurs in the gliotic white matter beyond the foci of necrosis in PVL, thus contributing to the white matter volume reduction. In a cohort of 17 control cases and 13 PVL cases with lesions of different chronological ages, diffuse axonal damage in PVL was detected by fractin in white matter sites surrounding and distant from acute and organizing foci of necrosis. Using β-APP, axonal spheroids were detected within necrotic foci in the acute and organizing (subacute) stages, a finding consistent with others. Interestingly, GAP-43 expression was also detected in spheroids in the necrotic foci, suggesting attempts at axonal regeneration. Thirty-one percent of the PVL cases had thalamic damage and 15% neuronal injury in the cerebral cortex overlying PVL. We conclude that diffuse axonal injury, as determined by apoptotic marker fractin, occurs in PVL and that its cause likely includes primary ischemia and trophic degeneration secondary to corticothalamic neuronal damage.

Interferon-γ Expression in Periventricular Leukomalacia in the Human Brain

Periventricular leukomalacia (PVL), the major lesion underlying cerebral palsy in survivors of prematurity, is characterized by focal periventricular necrosis and diffuse gliosis of immature cerebral white matter. Causal roles have been ascribed to hypoxiaischemia and maternal‐fetal infection, leading to cytokine responses, inflammation, and oligodendrocyte cell death. Because interferon‐γ (IFN‐γ) is directly toxic to immature oligodendrocytes, we tested the hypothesis that it is expressed in PVL (N=13) compared to age‐adjusted controls (N=31) using immunocytochemistry. In PVL, IFN‐γ immunopositive macrophages were clustered in necrotic foci, and IFN‐γ immunopositive reactive astrocytes were present throughout the surrounding white matter (WM). The difference in the number of IFN‐y immunopositive glial cells/high power field (IFN‐γ score, Grades 0–3) between PVL cases (age‐adjusted mean 2.59 ±0.25) and controls (age‐adjusted mean 1.39±0.16) was significant (p<0.001). In the gliotic WM, the IFN‐γ score correlated with markers for lipid peroxidation, but not nitrative stress. A subset of premyelinating (O4+) oligodendrocytes expressed IFN‐γ receptors in PVL and control cases, indicating that these cells are vulnerable to IFN‐γ toxicity via receptor‐mediated interactions. In PVL, IFN‐γ produced by macrophages and reactive astrocytes may play a role in cytokine‐induced toxicity to premyelinating oligodendrocytes as part of a cytokine response stimulated by ischemia and/or infection.

Thalamic Damage in Periventricular Leukomalacia: Novel Pathologic Observations Relevant to Cognitive Deficits in Survivors of Prematurity

Despite major advances in the long-term survival of premature infants, cognitive deficits occur in 30–50% of very preterm (<32 gestational weeks) survivors. Impaired working memory and attention despite average global intelligence are central to the academic difficulties of the survivors. Periventricular leukomalacia (PVL), characterized by periventricular necrosis and diffuse gliosis in the cerebral white matter, is the major brain pathology in preterm infants. We tested the novel hypothesis that pathology in thalamic nuclei critical for working memory and attention, i.e. mediodorsal nucleus and reticular nucleus, respectively, occurs in PVL. In 22 PVL cases (gestational age 32.5 ± 4.8 wk) and 16 non-PVL controls (36.7 ± 5.2 wk) who died within infancy, the incidence of thalamic pathology was significantly higher in PVL cases (59%; 13/22) compared with controls (19%; 3/16) (p = 0.01), with substantial involvement of the mediodorsal, and reticular nuclei in PVL. The prevention of thalamic damage may be required for the eradication of defects in survivors with PVL.

The Cerebral Cortex Overlying Periventricular Leukomalacia: Analysis of Pyramidal Neurons

The role of the cerebral cortex in the cognitive deficits in preterm survivors is poorly understood. Periventricular leukomalacia (PVL), the key feature of encephalopathy of prematurity, is characterized by periventricular necrotic foci and diffuse gliosis in the surrounding cerebral white matter. Here, we tested the hypothesis that reductions in the density of layer I neurons and/or pyramidal neurons in layers III and/or V are associated with PVL, indicating cortical pathology potentially associated with cognitive deficits in long‐term survivors. In controls (23 gestational weeks to 18 postnatal months) (n = 15), a lack of significant differences in pyramidal density among incipient Brodmann areas suggested that cytoarchitectonic differences across functional areas are not fully mature in the fetal and infant periods. There was a marked reduction (38%) in the density of layer V neurons in all areas sampled in the PVL cases (n = 17) compared to controls (n = 12) adjusted for postconceptional age at or greater than 30 weeks, when the six‐layer cortex is visually distinct (P < 0.024). This may reflect a dying‐back loss of somata complicating transection of layer V axons projecting through the necrosis in the underlying white matter. This study underscores the potential role of secondary cortical injury in the encephalopathy of prematurity.

Neuron deficit in the white matter and subplate in periventricular leukomalacia

The cellular basis of cognitive abnormalities in preterm infants with periventricular leukomalacia (PVL) is uncertain. One important possibility is that damage to white matter and subplate neurons that are critical to the formation of the cerebral cortex occurs in conjunction with oligodendrocyte and axonal injury in PVL. We tested the hypothesis that the overall density of neurons in the white matter and subplate region is significantly lower in PVL cases compared to non‐PVL controls.

TLR8: an innate immune receptor in brain, neurons and axons.

Toll-like receptors (TLRs) play essential roles in generating innate immune responses, and are evolutionarily conserved across species. In mammals, TLRs specifically recognize the conserved microbial structural motifs referred to as pathogen-associated molecular patterns (PAMPs). Ligand recognition by TLRs activates signaling cascades that culminate in proinflammatory cytokine production and eventual elimination of invading pathogens. Although TLRs in mammals are expressed predominantly in the immune system, certain TLRs with poorly characterized function are also found in neurons. We recently profiled TLR8 expression during mouse brain development and established its localization in neurons and axons. We uncovered a novel role for TLR8 as a suppressor of neurite outgrowth as well as an inducer of neuronal apoptosis, and found that TLR8 functions in neurons through an NF-κB-independent mechanism. These findings add a new layer of complexity for TLR signaling, and expand the realm of mammalian TLR function to the central nervous system (CNS) beyond the originally discovered immune context. Herein, we complement our earlier report with additional data, discuss their biological and mechanistic implications in CNS developmental and pathological processes, and thus further our perspective on TLR signaling and potential physiological roles in mammals.