Stephen A. Back

Human oligodendroglial development: Relationship to periventricular leukomalacia

Periventricular leukomalacia in the premature infant is a lesion of cerebral white matter with its greatest period of risk when white matter is immature, that is, when oligodendrocyte precursors are proliferating and differentiating, and before myelin sheaths are actively synthesized. Although the pathogenesis of perinatal cerebral white matter damage involves multiple factors, the correlation of the timing of the lesion with dominance of oligodendrocyte precursors in cerebral white matter suggests that intrinsic factors related to oligodendrocyte precursors are critical. Ischemia and infection have both been implicated as causes of perinatal white matter damage. Major mechanisms underlying oligodendrocyte injury in ischemia include glutamate toxicity, free-radical injury, and cytokine damage mediated by macrophages accompanying ischemia-induced inflammation. Factors related to a vulnerability of immature oligodendrocytes to ischemia potentially include a developmental lack of antioxidant enzymes to mediate oxidative stress. Cytokine-mediated injury to oligodendrocytes is also potentially important. A complete understanding of the role of immature white matter in the pathogenesis of periventricular leukomalacia is essential for developing strategies to prevent it.

Mature myelin basic protein-expressing oligodendrocytes are insensitive to kainate toxicity

We examined the vulnerability to excitotoxicity of rat oligodendrocytes in dissociated cell culture at different developmental stages. Mature oligodendrocytes that express myelin basic protein were resistant to excitotoxic injury produced by kainate, whereas earlier stages in the oligodendrocyte lineage were vulnerable to this insult. To test the hypothesis that the sensitivity of immature oligodendrocytes and the resistance of mature oligodendrocytes to kainate toxicity were due to differences in membrane responsiveness to kainate, we used whole‐cell patch‐clamp recording. Oligodendrocyte precursors in cultures vulnerable to kainate toxicity responded to 500 μM kainate with large inward currents, whereas mature myelin basic protein‐expressing oligodendrocytes in cultures resistant to kainate toxicity showed no clear response to application of this agonist. We assayed expression of glutamate receptor subunits (GluR) ‐2, ‐4, ‐6, ‐7, and KA2 using immunoblot analysis and found that expression of all of these glutamate receptors was significantly down‐regulated in mature oligodendrocytes. These results suggest a striking developmental regulation of glutamate receptors in oligodendrocytes and suggest that the vulnerability of oligodendrocytes to non‐ N‐methyl‐D‐aspartate receptor‐mediated excitotoxicity might be much greater in developing oligodendrocytes than after the completion of myelination.

Cystine Deprivation Induces Oligodendroglial Death: Rescue by Free Radical Scavengers and by a Diffusible Glial Factor

Abstract: In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1‐day‐old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC50 values for cystine for a 24‐h exposure ranged from 2 to 65 µM. After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 ± 13% of the control. Free radical scavengers (α‐tocopherol, ascorbate, idebenone, and N‐tert‐butyl‐α‐phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3‐day‐old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine‐depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine‐depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.

Cellular and molecular pathogenesis of periventricular white matter injury

Periventricular white matter injury has a high incidence, particularly in the premature infant. A prominent feature of the pathogenesis of this injury, which ultimately results in disrupted myelination of periventricular white matter tracts, is a loss of oligodendrocytes (OLs). The timing of injury corresponds to the period in white matter development when OL precursors predominate. This article focuses on current understanding of the cellular and molecular basis for the developmental vulnerability of OL precursors that may predispose to their loss in periventricular leukomalacia (PVL). Recent advances in the cellular neurobiology of OL development have permitted study of the processes that regulate survival of developing OLs. Several potentially complementary etiologies for the developmental vulnerability of OL precursors are reviewed: (1) free-radical–mediated toxicity in the setting of oxidative stress, (2) cytotoxic cytokines, and (3) a critical dependence on selected trophic factors during certain periods in OL development. Recent work indicates that these causes of cell death are mediated by a common mechanism involving apoptosis. Potential therapeutic interventions for interruption of the pathways mediating OL death are examined. MRDD Research Reviews 3:96–107, 1997.

Intracellular Redox State Determines Whether Nitric Oxide Is Toxic or Protective to Rat Oligodendrocytes in Culture

Abstract : We found that several nitric oxide donors had similar potency in killing mature and immature forms of oligodendrocytes (OLs). Because of the possibility of interaction of nitric oxide with intracellular thiols, we tested the effect of the nitrosonium ion donor S‐nitrosylglutathione (SNOG) in OL cultures in the setting of cystine deprivation, which has been shown to cause intracellular glutathione depletion. Surprisingly, the presence of 200 μM SNOG completely protected OLs against the toxicity of cystine depletion. This protection appeared to be due to nitric oxide, because it could be blocked by hemoglobin and potentiated by inclusion of superoxide dismutase. We tested the effect of three additional NO• donors and found that protection was not seen with diethylamine NONOate, a donor with a half‐life measured in minutes, but was seen with dipropylenetriamine NONOate and diethylaminetriamine NONOate, donors with half‐lives measured in hours. This need for donors with longer half‐lives for the protective effect suggested that NO• was required when intracellular thiol concentrations were falling, a process evolving over hours in medium depleted of cystine. These studies suggest a novel protective role for nitric oxide in oxidative stress injury and raise the possibility that intracerebral nitric oxide production might be a mechanism of defense against oxidative stress injury in OLs.

A new Alamar Blue viability assay to rapidly quantify oligodendrocyte death

We developed a rapid fluorometric viability assay for primary cultures of OL precursors (preOLs) or mature OLs that utilized the oxidation/reduction indicator dye Alamar Blue (AB). PreOLs had a lower rate of AB reduction than did mature OLs (0.02 +/- 0.01 units/min per cell versus 0.07 +/- 0.01). The assay was tested under two conditions toxic to preOLs: oxidative stress induced by glutathione depletion or kainate excitotoxicity. When glutathione was depleted by a 24-h exposure to cystine-depleted medium, the EC50 values for the dependence upon cystine for survival did not differ significantly when determined by AB reduction (2 +/- 2 microM), by the trypan blue exclusion method (3 +/- 3 microM) or by MTT histochemistry (1 +/- 0.4 microM). Quantification of preOL viability with AB was unaffected by the presence of free radical scavengers (alpha-tocopherol or idebenone) or the antioxidant enzymes Cu,Zn-superoxide dismutase and catalase. There was no difference in preOL viability as determined by AB or MTT after a 24-h exposure to kainate at concentrations up to 1 mM. AB offers a rapid objective measure of OL viability in primary culture and is a valid means to quantify OL death.

Strain-specific differences in perinatal rodent oligodendrocyte lineage progression and its correlation with human.

Progress in the development of rat models of human periventricular white matter injury (WMI) has been hampered by uncertainty about the developmental window in different rodent strains that coincides with cerebral white matter development in human premature infants. To define strain-specific differences in rat cerebral white matter maturation, we analyzed oligodendrocyte (OL) lineage maturation between postnatal days (P)2 and P14 in three widely studied strains of rat: Sprague-Dawley, Long-Evans and Wistar (W). We previously reported that late OL progenitors (preOL) are the major vulnerable cell type in human periventricular WMI. Strain-specific differences in preOL maturation were found at P2, such that the W rat had the highest percentage and density of preOL relative to the other strains. Overall, at P2, the state of OL maturation was similar to preterm human cerebral white matter. However, by P5, all three strains displayed a similar magnitude and extent of OL maturation that persisted with progressive myelination between P7 and P14. PreOL were the predominant OL lineage stage present in the cerebral cortex through P14, and thus OL lineage maturation occurred latter than in white matter. The hippocampus also displayed a later onset of preOL maturation in all three strains, such that OL lineage maturation and early myelination was not observed to occur until about P14. This timing of preOL maturation in rat cortical gray matter coincided with a similar timing in human cerebral cortex, where preOL also predominated until at least 8 months after full-term birth. These studies support that strain-specific differences in OL lineage immaturity were present in the early perinatal period at about P2, and they define a narrow window of preterm equivalence with human that diminishes by P5. Later developmental onset of preOL maturation in both cerebral cortex and hippocampus coincides with an extended window of potential vulnerability of the OL lineage to hypoxia-ischemia in these gray matter regions.

Approaches to the Study of Diseases Involving Oligodendroglial Death

Oligodendrocytes (OLs) are the myelin-producing cells of the central nervous system (CNS). Myelin is the compact, multilamellar, lipid-rich sheath that envelops axons. As a result of this membrane insulation, the rate of conduction of nerve action potentials down an axon is maximized while energy and space demands within the CNS are minimized. Disorders of myelin (i.e., white matter disorders) result from a variety of causes (see representative examples in Table 1). Extensive nervous system demyelination often has devastating consequences, including paralysis, dementia, or coma. These clinical sequelae reflect the marked reduction in the ability of the nervous system to conduct nerve impulses at a normal rate. Consider, for example, one form of the developmental disorder cerebral palsy (CP), in which injury to perinatal cerebral white matter results in decreased myelination of regions subserving such neurologic functions as motor control and vision. Around 5–15% of the nearly 50,000 preterm infants born in the US each year will acquire CP related to white matter damage, and manifesting as spastic motor deficits, and another 25–50% will show evidence of learning disabilities (1). The estimated economic cost to society of CP is in excess of five billion dollars annually.

Encephalopathy of Prematurity: Pathophysiology

The neuropathology of the encephalopathy of prematurity (see Chapter 14) includes injury to cerebral white matter and, in a major proportion of infants, a variety of neuronal-axonal disturbances. The encephalopathy includes a broad spectrum of initiating destructive events or disturbances in cellular maturation that are followed by widespread disruptions in cerebral white and gray matter growth and neuronal connectivity. This chapter emphasizes the pathophysiology of the most consistent and well-defined feature of the encephalopathy, injury to the cerebral white matter.