Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants.

OBJECTIVE: The major form of magnetic resonance imaging-defined white matter injury (WMI) comprises diffuse lesions where the burden of small necrotic foci (microscopic necrosis) is poorly defined. We hypothesized that myelination failure associated with diffuse WMI involves an aberrant injury response linked to arrested preoligodendrocyte (preOL) maturation in reactive astrocyte-rich lesions. METHODS: A retrospective autopsy series (1983-2000) was selected for cases with diffuse WMI and analyzed relative to prospectively collected contemporary cases (2003-2010). Controls were age- and region-matched to address regional variation in preOL maturation. Successive oligodendrocyte stages were analyzed with lineage-specific markers. Microscopic necrosis was quantified with microglial markers. Axon injury markers defined the burden of axonopathy. Extracellular matrix remodeling was defined by detection of hyaluronic acid (HA), an inhibitor of preOL maturation, and the HA receptor, CD44. RESULTS: In the contemporary case series, diffuse WMI was accompanied by a significant reduction in the burden of microscopic necrosis and axonopathy. Diffuse astrogliosis extended into the lesion surround with elevated HA and astrocyte-expressed CD44. The total population of OL lineage stages was significantly increased in lesions. This increase coincided with significant expansion of the preOL pool. INTERPRETATION: Although these data confirm that microscopic necrosis occurs in contemporary cases, the markedly decreased burden supports that it does not contribute substantially to myelination failure. The primary mechanism of myelination failure involves a disrupted cellular response whereby preOLs fail to differentiate in diffuse astrogliotic lesions. PreOL maturation arrest converts chronic WMI to a more immature state related to the burden of astrogliosis.

Hyaluronan accumulates in demyelinated lesions and inhibits oligodendrocyte progenitor maturation.

Demyelination is the hallmark of numerous neurodegenerative conditions, including multiple sclerosis. Oligodendrocyte progenitors (OPCs), which normally mature into myelin-forming oligodendrocytes, are typically present around demyelinated lesions but do not remyelinate affected axons. Here, we find that the glycosaminoglycan hyaluronan accumulates in demyelinated lesions from individuals with multiple sclerosis and in mice with experimental autoimmune encephalomyelitis. A high molecular weight (HMW) form of hyaluronan synthesized by astrocytes accumulates in chronic demyelinated lesions. This form of hyaluronan inhibits remyelination after lysolecithin-induced white matter demyelination. OPCs accrue and do not mature into myelin-forming cells in demyelinating lesions where HMW hyaluronan is present. Furthermore, the addition of HMW hyaluronan to OPC cultures reversibly inhibits progenitor-cell maturation, whereas degrading hyaluronan in astrocyte-OPC cocultures promotes oligodendrocyte maturation. HMW hyaluronan may therefore contribute substantially to remyelination failure by preventing the maturation of OPCs that are recruited to demyelinating lesions.

Arrested oligodendrocyte lineage maturation in chronic perinatal white matter injury.

OBJECTIVE: Abnormal myelination is a major pathological sequela of chronic periventricular white matter injury in survivors of premature birth. We tested the hypothesis that myelination failure in chronic hypoxia-ischemia-induced periventricular white matter injury is related to persistent depletion of the oligodendrocyte (OL) precursor pool required to generate mature myelinating OLs. METHODS: A neonatal rat model of hypoxia-ischemia was used where acute degeneration of late OL progenitors (preOLs) occurs via a mostly caspase-independent mechanism. The fate of OL lineage cells in chronic cerebral lesions was defined with OL lineage-specific markers. RESULTS: Acute caspase-3-independent preOL degeneration from hypoxia-ischemia was significantly augmented by delayed preOL death that was caspase-3-dependent. Degeneration of preOLs was offset by a robust regenerative response that resulted in a several-fold expansion in the pool of surviving preOLs in chronic lesions. However, these preOLs displayed persistent maturation arrest with failure to differentiate and generate myelin. When preOL-rich chronic lesions sustained recurrent hypoxia-ischemia at a time in development when white matter is normally resistant to injury, an approximately 10-fold increase in caspase-dependent preOL degeneration occurred relative to lesions caused by a single episode of hypoxia-ischemia. INTERPRETATION: The mechanism of myelination failure in chronic white matter lesions is related to a combination of delayed preOL degeneration and preOL maturation arrest. The persistence of a susceptible population of preOLs renders chronic white matter lesions markedly more vulnerable to recurrent hypoxia-ischemia. These data suggest that preOL maturation arrest may predispose to more severe white matter injury in preterm survivors that sustain recurrent hypoxia-ischemia.

Cerebral blood flow heterogeneity in preterm sheep: lack of physiologic support for vascular boundary zones in fetal cerebral white matter.

Periventricular white matter (PVWM) injury is the leading cause of neurologic disability in survivors of prematurity. To address the role of ischemia in PVWM and cerebral cortical injury, we hypothesized that immaturity of spatially distal vascular 'end zones' or 'border zones' predisposes PVWM to greater decreases in cerebral blood flow (CBF) than more proximal structures. We quantified regional CBF with fluorescently labeled microspheres in 0.65 gestation fetal sheep in histopathologically defined three-dimensional regions by post hoc digital dissection and coregistration algorithms. Basal flow in PVWM was significantly lower than in gyral white matter and cortex, but was equivalent in superficial, middle, and deep PVWM. Absolute and relative CBF (expressed as percentage of basal) did not differ significantly during ischemia or reperfusion between PVWM, gyral white matter, or cortex. Moreover, CBF during ischemia-reperfusion was equivalent in three adjacent PVWM levels and was not consistent with the magnitude of severity of PVWM injury, defined by TUNEL (terminal deoxynucleotidyltransferase-mediated dUPT nick end labeling) staining. However, the magnitude of ischemia was predicted by the severity of discrete cortical lesions. Hence, unlike cerebral cortex, unique CBF disturbances did not account for the distribution of PVWM injury. Previously defined cellular maturational factors, thus, appear to have a greater influence on PVWM vulnerability to ischemic injury than the presence of immature vascular boundary zones.

Spatial heterogeneity in oligodendrocyte lineage maturation and not cerebral blood flow predicts fetal ovine periventricular white matter injury.

Although periventricular white matter injury (PWMI) is the leading cause of chronic neurological disability and cerebral palsy in survivors of premature birth, the cellular-molecular mechanisms by which ischemia-reperfusion contributes to the pathogenesis of PWMI are not well defined. To define pathophysiologic relationships among ischemia, acute cerebral white matter damage, and vulnerable target populations, we used a global cerebral ischemia-reperfusion model in the instrumented 0.65 gestation fetal sheep. We developed a novel method to make repeated measurements of cerebral blood flow using fluorescently labeled microspheres to resolve the spatial heterogeneity of flow in situ in three-dimensional space. Basal flow in the periventricular white matter (PVWM) was significantly lower than in the cerebral cortex. During global cerebral ischemia induced by carotid occlusion, flow to all regions was reduced by nearly 90%. Ischemia of 30 or 37 min duration generated selective graded injury to frontal and parietal PVWM, two regions of predilection for human PWMI. Injury was proportional to the duration of ischemia and increased markedly with 45 min of ischemia to extensively damage cortical and subcortical gray matter. Surprisingly, the distribution of PVWM damage was not uniform and not explained by heterogeneity in the degree of white matter ischemia. Rather, the extent of white matter damage coincided with the presence of a susceptible population of late oligodendrocyte progenitors. These data support that although ischemia is necessary to generate PWMI, the presence of susceptible populations of oligodendrocyte progenitors underlies regional predilection to injury.

Hypoxia-ischemia preferentially triggers glutamate depletion from oligodendroglia and axons in perinatal cerebral white matter.

Ischemia is implicated in periventricular white matter injury (PWMI), a lesion associated with cerebral palsy. PWMI features selective damage to early cells of the oligodendrocyte lineage, a phenomenon associated with glutamate receptor activation. We have investigated the distribution of glutamate in rat periventricular white matter at post-natal day 7. Immuno-electron microcopy was used to identify O4(+) oligodendroglia in control rats, and a similar approach was employed to stain glutamate in these cells before and after 90 mins of hypoxia-ischemia. This relatively brief period of hypoxia-ischemia produced mild cell injury, corresponding to the early stages of PWMI. Glutamate-like reactivity was higher in oligodendrocytes than in other cell types (2.13+/-0.25 counts/microm(2)), and declined significantly during hypoxia-ischemia (0.93+/-0.15 counts/microm(2): P<0.001). Astrocytes had lower glutamate levels (0.7+/-0.07 counts/microm(2)), and showed a relatively small decline during hypoxia-ischemia. Axonal regions contained high levels of glutamate (1.84+/-0.20 counts/microm(2)), much of which was lost during hypoxia-ischemia (0.72+/-0.20 counts/microm(2): P>0.001). These findings suggest that oligodendroglia and axons are the major source of extracellular glutamate in developing white matter during hypoxia-ischemia, and that astrocytes fail to accumulate the glutamate lost from these sources. We also examined glutamate levels in the choroid plexus. Control glutamate levels were high in both choroid epithelial (1.90+/-0.20 counts/microm(2)), and ependymal cells (2.20+/-0.28 counts/microm(2)), and hypoxia-ischemia produced a large fall in ependymal glutamate (0.97+/-0.08 counts/microm(2): P>0.001). The ependymal cells were damaged by the insult and represent a further potential source of glutamate during ischemia.

Developmental changes in diffusion anisotropy coincide with immature oligodendrocyte progression and maturation of compound action potential.

Disruption of oligodendrocyte lineage progression is implicated in the white-matter injury that occurs in cerebral palsy. We have previously published a model in rabbits consistent with cerebral palsy. Little is known of normal white-matter development in perinatal rabbits. Using a multidimensional approach, we defined the relationship of oligodendrocyte lineage progression and functional maturation of axons to structural development of selected cerebral white-matter tracts as determined by diffusion tensor imaging (DTI). Immunohistochemical studies showed that late oligodendrocyte progenitors appear at gestational age 22 [embryonic day 22 (E22)], whereas immature oligodendrocytes appear at E25, and both increase rapidly with time (approximately 13 cells/mm2/d) until the onset of myelination. Myelination began at postnatal day 5 (P5) (E36) in the internal capsule (IC) and at P11 in the medial corpus callosum (CC), as determined by localization of sodium channels and myelin basic protein. DTI of the CC and IC showed that fractional anisotropy (FA) increased rapidly between E25 and P1 (E32) (11% per day) and plateaued (<5% per day) after the onset of myelination. Postnatal maturation of the compound action potential (CAP) showed a developmental pattern similar to FA, with a rapid rise between E29 and P5 (in the CC, 18% per day) and a slower rise from P5 to P11 (in the CC, <5% per day). The development of immature oligodendrocytes after E29 coincides with changes in FA and CAP area in both the CC and IC. These findings suggest that developmental expansion of immature oligodendrocytes during the premyelination period may be important in defining structural and functional maturation of the white matter.

Preterm fetal hypoxia-ischemia causes hypertonia and motor deficits in the neonatal rabbit: a model for human cerebral palsy?

Prenatal hypoxia-ischemia to the developing brain has been strongly implicated in the subsequent development of the hypertonic motor deficits of cerebral palsy (CP) in premature and full-term infants who present with neonatal encephalopathy. Despite the enormous impact of CP, there is no animal model that reproduces the hypertonia and motor disturbances of this disorder. We report a rabbit model of in utero placental insufficiency, in which hypertonia is accompanied by marked abnormalities in motor control. Preterm fetuses (67-70% gestation) were subjected to sustained global hypoxia. The dams survived and gave spontaneous birth. At postnatal day 1, the pups that survived were subjected to a battery of neurobehavioral tests developed specifically for these animals, and the tests were videotaped and scored in a masked manner. Newborn pups of hypoxic groups displayed significant impairment in multiple tests of spontaneous locomotion, reflex motor activity, and the coordination of suck and swallow. Increased tone of the limbs at rest and with active flexion and extension were observed in the survivors of the preterm insult. Histopathological studies identified a distinct pattern of acute injury to subcortical motor pathways that involved the basal ganglia and thalamus. Persistent injury to the caudate putamen and thalamus at P1 was significantly correlated with hypertonic motor deficits in the hypoxic group. Antenatal hypoxia-ischemia at preterm gestation results in hypertonia and abnormalities in motor control. These findings provide a unique behavioral model to define mechanisms and sequelae of perinatal brain injury from antenatal hypoxia-ischemia.

Selective vulnerability of late oligodendrocyte progenitors to hypoxia-ischemia.

In the premature infant, hypoxic-ischemic damage to the cerebral white matter [periventricular leukomalacia (PVL)] is a common and leading cause of brain injury that often results in chronic neurologic disability from cerebral palsy. The cellular basis for the propensity of white matter injury to occur in the developing brain and the greater resistance of the adult white matter to similar injury remains unknown. By using a neonatal rat model of hypoxic-ischemic injury, we found that the mechanism of perinatal white matter injury involved maturation-dependent vulnerability in the oligodendroctye (OL) lineage. The timing of appearance of late OL progenitors was the major developmental factor that accounted for the susceptibility of the neonatal white matter to injury. Late OL progenitors were the major OL lineage stage killed by apoptosis, whereas early OL progenitors and more mature OLs were highly resistant. The density of pyknotic late OL progenitors was significantly increased in the ischemic hemisphere (67 +/- 31 cells/mm2) versus the control hemisphere (2.2 +/- 0.4 cells/mm2; mean +/- SEM; p = 0.05), which resulted in the death of 72 +/- 6% of this OL stage. Surviving late OL progenitors displayed a reactive response in which an increase in cell density was accompanied by accelerated maturation to a P27/kip1-positive oligodendrocyte. Because we showed recently that late OL progenitors populate human cerebral white matter during the high risk period for PVL (Back et al., 2001), maturation-dependent vulnerability of OL progenitors to hypoxia-ischemia may underlie the selective vulnerability to PVL of the white matter in the premature infant.

Arrested oligodendrocyte lineage progression during human cerebral white matter development: dissociation between the timing of progenitor differentiation and myelinogenesis.

Immature oligodendrocytes (OLs) derive from a large pool of late OL progenitors that populate human cerebral white matter throughout the latter half of gestation. We recently reported that a minor population of immature OLs are present in human cerebral white matter for at least 3 months before these cells commit to myelinogenesis around 30 wk postconceptional age. Since this finding supports dissociation between the events that regulate human immature OL maturation and their commitment to myelinogenesis, we characterized here the cellular sequence of events that characterize immature OLs during the transition from a premyelinating to a myelinating state. Commitment of immature OLs to myelinogenesis in human cerebral white matter correlated with the longitudinal extension of specialized processes, designated "pioneer processes," that made multiple types of apparent contacts with axons. This event coincided with the appearance of 3 distinct populations of sheaths that varied in their labeling for myelin basic protein (MBP). Since few axons initially labeled for MBP, this supported the occurrence in vivo of O4-negative, O1-positive premyelin sheaths that precede MBP-positive compacted myelin. These observations identify 3 sequential stages of early myelinogenesis: 1) the initial ensheathment of axons by premyelin sheaths generated by immature OLs; 2) the initial insertion of MBP into transitional sheaths; and 3) the generation of MBP-rich mature myelin.

Timing of appearance of late oligodendrocyte progenitors coincides with enhanced susceptibility of preterm rabbit cerebral white matter to hypoxia-ischemia.

Emerging evidence supports that premature infants are susceptible to both cerebral white and gray matter injury. In a fetal rabbit model of placental insufficiency, preterm rabbits at embryonic day 22 (E22) exhibited histologic evidence of gray matter injury but minimal white matter injury after global hypoxia-ischemia (H-I). We hypothesized that the dissociation between susceptibility to gray and white matter injury at E22 was related to the timing of appearance of late oligodendrocyte progenitors (preOLs) that are particularly vulnerable in preterm human white matter lesions. During normal rabbit oligodendrocyte (OL) lineage progression, early OL progenitors predominated at E22. PreOL density increased between E24 and E25 in major forebrain white matter tracts. After H-I at E22 and E25, we observed a similar magnitude of cerebral H-I, assessed by cortical microvascular blood flow, and gray matter injury, assessed by caspase activation. However, the increased preOL density at E25 was accompanied by a significant increase in acute white matter injury after H-I that coincided with enhanced preOL degeneration. At E29, significant white matter atrophy developed after H-I at E25 but not E22. Thus, the timing of appearance of preOLs coincided with onset of a developmental window of enhanced white but not gray matter susceptibility to H-I.

Protective effects of caffeine on chronic hypoxia-induced perinatal white matter injury.

OBJECTIVE: Periventricular white matter injury (PWMI) is the major cause of cerebral palsy and cognitive impairment in prematurely born infants. PWMI is characterized by reductions in cerebral myelination and cerebrocortical volumes and is associated with secondary ventriculomegaly. In neonatal rodents, these features of PWMI can be induced by rearing in chronic hypoxia or by activation of A1 adenosine receptors. We determined: (1) whether altered maturation or development of one or more oligodendrocyte (OL) lineage stages plays a role in the pathogenesis of the myelination disturbances associated with exposure to chronic hypoxia, and (2) whether blockade of A1 adenosine receptor action with the adenosine antagonist caffeine can prevent hypoxia-induced white matter injury. METHODS: Ventriculomegaly and reduced cerebral myelination were generated in mice reared in hypoxia (10% oxygen) from postnatal days 3 (P3) through 12. RESULTS: Hypomyelination was related to abnormal OL lineage progression and a reduction in the OL progenitor pool. Myelination was enhanced and ventriculomegaly reduced in hypoxia-exposed neonatal pups treated with caffeine from P3 to P12. INTERPRETATION: These observations support that hypoxia inhibits OL maturation and that caffeine administration during early postnatal development may have utility in the prevention of PWMI.

Selective vulnerability of preterm white matter to oxidative damage defined by F2-isoprostanes.

Periventricular white matter injury (PWMI) is the leading cause of cerebral palsy and chronic neurological disability in survivors of prematurity. Despite the large number of affected children, the pathogenetic mechanisms related to PWMI remain controversial. Through studies of 33 human autopsy brains, we determined that early PWMI was related to oxidative damage that particularly targeted the oligodendrocyte lineage, whereas other neuronal and glial cell types were markedly more resistant. F(2)-isoprostanes, an arachidinate metabolite/lipid peroxidation marker of oxidative damage, were significantly increased in early PWMI lesions but not in cerebral cortex. That deleterious lipid peroxidation accompanied early PWMI was supported by similar increases in F(2)-isoprostanes levels in the cerebral cortex from term infants with hypoxic-ischemic cortical injury. Detection of F(4)-neuroprostanes, a neuronal-specific oxidative damage marker, confirmed that neuroaxonal elements were resistant to injury in cerebral cortex and white matter. Significant protein nitration was not detected in PWMI lesions by 3-nitrotyrosine staining. Significant cellular degeneration was confirmed in early PWMI lesions by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and a marked depletion of oligodendrocyte progenitors of 71 +/- 8%. Hence, the predilection of preterm infants for PWMI is related to selective lipid peroxidation-mediated injury of cerebral white matter and targeted death of oligodendrocyte progenitors.