Local uncoupling of the cerebrovascular and metabolic responses to somatosensory stimulation after neuronal nitric oxide synthase inhibition

In order to investigate the remyelinating potential of mature oligodendrocytes in vivo, we have developed a model of demyelination in the adult rat spinal cord in which some oligodendrocytes survive demyelination. A single intraspinal injection of complement proteins plus antibodies to galactocerebroside (the major myelin sphingolipid) resulted in demyelination followed by oligodendrocyte remyelination. Remyelination was absent when the spinal cord was exposed to 40 Grays of x-irradiation prior to demyelination, a procedure that kills dividing cells. Quantitative Rip immunohistochemical analysis revealed a similar density of surviving oligodendrocytes in x-irradiated and nonirradiated lesions 3 days after demyelination. Rip and bromodeoxyuridine double immunohistochemical analysis of demyelinated lesions indicated that Rip+ oligodendrocytes did not divide as an acute response to demyelination. Oligodendrocytes were also identified by Rip immunostaining and electron microscopy at late time points (3 weeks) within x-irradiated areas of demyelination. These oligodendrocytes extended processes that engaged axons, and on occasion formed myelin membranes, but did not lay down new myelin sheaths. These studies demonstrate that (a) oligodendrocytes that survive within a region of demyelination are not induced to divide in the presence of demyelinated axons, and (b) fully-differentiated oligodendrocytes are therefore postmitotic and do not contribute to remyelination in the adult CNS.     

Teaching multiskilling in dietetics education

The reaction of oligodendrocytes in response to traumatic injury of the CNS are poorly understood. In the present report we studied changes in the expression of a major constituent of CNS myelin, myelin basic protein (MBP), by immunohistochemistry and in situ hybridization from 6 h up to 2 weeks following partial transection of the spinal cord in adult rats. MBP immunohistochemistry showed degeneration of myelin at the lesion center and signs of myelin breakdown in necrotic foci in the dorsal and ventral funiculi proximal and distal to the lesion. In situ hybridization revealed that mRNA for MBP was downregulated at the local lesion site within the first day following injury, probably reflecting oligodendrocytes to undergo cell death. From 2 days on, however, MBP mRNA was conspicuously upregulated at the border of the lesion area. This "reactive" response of surviving oligodendrocytes, as indicated by increased levels of MBP mRNA, peaked around 8 days. At this time, oligodendrocytes displaying strong MBP in situ signal formed stripe-like structures which were oriented radially toward the lesion center and arranged in parallel to neurofilament-positive axons. At around 2 weeks post-injury, MBP mRNA at the border of the lesion area was again downregulated to levels comparable to uninjured controls. These results show that traumatic injury of the spinal cord induces a "reactive" response of surviving oligodendrocytes adjacent to lesion sites. This response might represent an important component of local repair mechanisms.     

Density-dependent feedback inhibition of oligodendrocyte precursor expansion

The myelin sheath in the vertebrate CNS is formed by oligodendrocytes. The number of oligodendrocytes in a mature axon tract must be sufficient to myelinate all appropriate axons. How the number of oligodendrocytes is matched to axonal requirements and whether such matching involves axon-oligodendrocyte signaling or intrinsic oligodendrocyte self-regulation are not clear. Using a combination of in vitro analyses, we demonstrate that oligodendrocyte precursors closely regulate their numbers through interactions between adjacent precursors. In low-density rat spinal cord cultures, the number of oligodendrocyte lineage cells increases rapidly. The addition of large numbers of oligodendrocyte precursors substantially reduces precursor expansion and results in a normalization of oligodendrocyte lineage cell numbers in the cultures over time. Thus, the number of oligodendrocyte lineage cells that develop appears dependent on the density of oligodendrocyte lineage cells. This normalization of cell number is reflected in assays of clonal potential and proliferation. For example, precursors gave rise to fewer progeny and proliferated less at high density. Reduced precursor expansion at high density was not attributable to the depletion of growth factors. Cocultures of high and low densities did not inhibit precursor expansion in low-density cultures, suggesting the requirement for local cell-cell interactions. The inhibition of precursor expansion was cell-type-specific and dependent on the presence of oligodendrocyte lineage cells. We propose that this density-dependent feedback inhibition of oligodendrocyte precursor expansion may play a primary role in regulating the number of oligodendrocytes in the developing spinal cord.     

Ex vivo and in vitro testis and ovary explants: utility for identifying steroid biosynthesis inhibitors and comparison to a Tier I screening battery

Retinal ganglion cell (RGC) axons in lizards (reptiles) were found to regenerate after optic nerve injury. To determine whether regeneration occurs because the visual pathway has growth-supporting glia cells or whether RGC axons regrow despite the presence of neurite growth-inhibitory components, the substrate properties of lizard optic nerve myelin and of oligodendrocytes were analyzed in vitro, using rat dorsal root ganglion (DRG) neurons. In addition, the response of lizard RGC axons upon contact with rat and reptilian oligodendrocytes or with myelin proteins from the mammalian central nervous system (CNS) was monitored. Lizard optic nerve myelin inhibited extension of rat DRG neurites, and lizard oligodendrocytes elicited DRG growth cone collapse. Both effects were partially reversed by antibody IN-1 against mammalian 35/250 kD neurite growth inhibitors, and IN-1 stained myelinated fiber tracts in the lizard CNS. However, lizard RGC growth cones grew freely across oligodendrocytes from the rat and the reptilian CNS. Mammalian CNS myelin proteins reconstituted into liposomes and added to elongating lizard RGC axons caused at most a transient collapse reaction. Growth cones always recovered within an hour and regrew. Thus, lizard CNS myelin and oligodendrocytes possess nonpermissive substrate properties for DRG neurons--like corresponding structures and cells in the mammalian CNS, including mammalian-like neurite growth inhibitors. Lizard RGC axons, however, appear to be far less sensitive to these inhibitory substrate components and therefore may be able to regenerate through the visual pathway despite the presence of myelin and oligodendrocytes that block growth of DRG neurites.     

Purification of the insecticidal toxin in crystals of Bacillus thuringiensis

Newly transected or denervated segments of isogeneic rat tibial nerve were implanted into the rat midbrain and sampled at weekly intervals up to 6 weeks post-operation. By 3 weeks, the peripheral nervous system (PNS) grafts were well-vascularized and contained Schwann cells, axons associated with Schwann cell processes, and macrophages. From 3 to 6 weeks, many axons within both the fresh and predegenerated grafts were myelinated by Schwann cells. The nerve fiber arrangement within the implant was similar to that of regenerating peripheral nerve in situ. The central nervous system (CNS) border of the implant was clearly demarcated by a rim of astrocytes behind which was a layer of regenerating oligodendrocytes and axons. Extending from the CNS margin were radial bridges of astroglial tissue which apprarently guided regenerating axons into the implant. Between the CNS and the PNS implant, abundant collagen deposition was present. The findings suggest that regenerating CNS axons grow via astroglial bridges into transplanted PNS tissue and are capable of stimulating the implanted Schwann cells to form myelin. Even Schwann cells deprived of axonal contact for prolonged periods were still capable of PNS myelin formation.     

Induction of metallothionein in astrocytes and microglia in the spinal cord from the myelin-deficient jimpy mouse

Jimpy is a shortened life-span murine mutant whose genetic disorder results in severe pathological alterations in the CNS, including hypomyelination, oligodendrocyte death and strong astroglial and microglial reaction. The knowledge of metallothionein (MT) regulation in the CNS and especially of MT presence in specific glial cell types under pathological conditions is scarce. In the present study, immunocytochemical detection of MT-I + II has been performed in spinal cord sections from 10-12- and 20-22-day-old jimpy and normal animals. The identification of MT-positive glial cells was achieved through double labeling combining MT immunocytochemistry and selective markers for oligodendrocytes, astrocytes and microglia. MT was found in glial cells and was present in the spinal cord of jimpy and normal mice at both ages, but there were remarkable differences in MT expression and in the nature of MT-positive glial cells depending on the type of mouse. The number of MT-positive cells was higher in jimpy than in normal spinal cords. This was apparent in all spinal cord areas, although it was more pronounced in white than in the gray matter and at 20-22 days than at 10-12 days. The mean number of MT-positive glia in the jimpy white matter was 1.9-fold (10-12 days) and 2.4-fold (20-22 days) higher than in the normal one. Astrocytes were the only parenchymal glial cells that were positively identified as MT-producing cells in normal animals. Interestingly, MT in the jimpy spinal cord was localized not only in astrocytes but also in microglial cells. The occurrence of MT induction in relation to reactive astrocytes and microglia, and its role in neuropathological conditions is discussed.     

Methodological issues for biomarkers and intermediate outcomes in cohort studies

Jimpy is a shortened life-span murine mutant showing recessive sex-linked inheritance. The genetic defect consists of a point mutation in the PLP gene and produces a severe CNS myelin deficiency that is associated with a variety of complex abnormalities affecting all glial populations. The myelin deficiency is primarily due to a failure to produce the normal amount of myelin during development. However, myelin destruction and oligodendrocyte death also account for the drastic myelin deficit observed in jimpy. The oligodendroglial cell line shows complex abnormalities in its differentiation pattern, including the degeneration of oligodendrocytes through an apoptotic mechanism. Oligodendrocytes seem to be the most likely candidate to be primarily altered in a disorder affecting myelination, but disturbances affecting astrocytes and microglia are also remarkable and may have a crucial significance in the development of the jimpy disorder. In fact, the jimpy phenotype may not be attributed to a defect in a single cell but rather to a deficiency in the normal relations between glial cells. Evidences from a variety of sources indicate that the jimpy mutant could be a model for disturbed glial development in the CNS. The accurate knowledge of the significance of PLP and its regulation during development must be of vital importance in order to understand glial abnormalities in jimpy.     

Effect of static compression on proteoglycan biosynthesis by chondrocytes transplanted to articular cartilage in vitro

Mutant mice that lack either protein zero (P0) or connexin 32 (Cx32) were generated previously to investigate the function of these myelin proteins in peripheral nerves and to assess the value of these mice as animal models for hereditary human peripheral neuropathies. Mice that are completely devoid of P0 expression (P0(+/0)) show a complex phenotype that is characterized by hypomyelination, compromised myelin compaction, and degeneration of myelin and axons early in life. In contrast, young mouse mutants that have retained one wild-type allele of the P0 gene (P0(+/0)) reveal morphologically normal myelin but start to develop signs of demyelination and remyelination at 4 months of age. A similar late-onset myelin deficiency was observed in Cx32-deficient mice (Cx32(0/0)). We have now generated mice deficient for Cx32 and P0. In animals that lack both proteins (Cx32(0/0)/P0(0/0), the phenotype is morphologically identical to mice that solely lack P0. Animals that lack Cx32 and carry one functional P0 allele (Cx32(0/0/P0(+/0)) revealed demyelination and remyelination as evidenced by thin myelin and Schwann cell onion bulb formation already at the age of 4 weeks, a time point when no pathology was observed in the single mutants. These morphological deficits were also more prominent in 4-month-old Cx32(0/0)/P0(+/0)animals compared to the single mutants. Our data support the view that Cx32 and P0 are crucial molecules for the maintenance of myelin. Furthermore, the function of Cx32 in the peripheral nervous system appears to be largely dispensable when myelin compaction is impaired.     

Stored blood--an effective immunosuppressive method for transplantation of kidneys from unrelated donors. An 11-year follow-up

The onset of myelination in the embryonic chick spinal cord begins on embryonic day (E) 12 or E13 of the 21 day in ovo developmental period. This event coincides with a loss of functional axonal regeneration following complete transection of the thoracic spinal cord. In this study, we have characterised an immunological method for delaying the developmental onset of myelination in vivo until later stages of development (developmental myelin-suppression). A single injection of heterologous or homologous serum complement proteins plus myelin-specific, complement-binding antibodies into the spinal cord prior to E13 delayed the onset of myelination until E17. The state of spinal cord myelin was assessed with immunohistochemical, histological and ultrastructural techniques. Northern blot analysis indicated that myelin basic protein mRNA was not down-regulated in myelin-suppressed spinal cords, which suggests that oligodendrocytes survived developmental myelin-suppression. Glial fibrillary acidic protein immunostaining of normal and treated tissue indicated that myelin-suppression did not alter the resident astrocyte population of the spinal cord or elicit astrogliosis. Immunostaining with microtubule-associated protein-2 and thionine staining of normal and myelin-suppressed tissue further indicated that the neuronal architecture was unaffected by the immunological protocol.     

Failure of spinal cord oligodendrocyte development in mice lacking neuregulin

Oligodendrocytes develop from a subpopulation of precursor cells within the ventral ventricular zone of the spinal cord. The molecular cues that direct this spatially and temporally restricted event seem to originate in part from structures ventral to and within the spinal cord. Here, we present evidence that the family of ligands termed neuregulins are necessary for the normal generation of mouse spinal cord oligodendrocytes. Oligodendrocytes mature in spinal cord explants from wild-type mice and mice heterozygotic for a null mutation in the neuregulin gene (NRG +/-) in a temporal sequence of developmental events that replicates that observed in vivo. However, in spinal cord explants derived from mice lacking neuregulin (NRG -/-), oligodendrocytes fail to develop. Addition of recombinant neuregulin to spinal cord explants from NRG -/- mice rescues oligodendrocyte development. In wild-type spinal cord explants, inhibitors of neuregulin mimic the inhibition of oligodendrocyte development that occurs in NRG -/- explants. In embryonic mouse spinal cord, neuregulins are present in motor neurons and the ventral ventricular zone where they likely exert their influence on early oligodendrocyte precursor cells.     

Apoptosis of microglia and oligodendrocytes after spinal cord contusion in rats

Following spinal cord contusion in the rat, apoptosis has been observed in the white matter for long distances remote from the center of the lesion and is primarily associated with degenerating fiber tracts. We have previously reported that many of the apoptotic cells are oligodendrocytes. Here we show that the oligodendrocyte death is maximal at 8 days postinjury and suggest that loss of oligodendrocytes may result in demyelination of axons that have survived the initial trauma. There are two mechanisms that may account for the observed oligodendrocyte apoptosis. The apoptotic cell death may result from the loss of trophic support after axonal degeneration or it may be the consequence of microglial activation. The hypothesis that oligodendrocyte apoptosis is secondary to microglial activation is supported by our observations of microglia with an activated morphology in the same regions as apoptosis and apparent contact between some of the apoptotic oligodendrocytes and microglial processes. In addition to oligodendrocyte apoptosis, a subpopulation of microglia appears to be susceptible to apoptotic cell death as well, as evidenced by the presence of apoptotic bodies in OX42 immunopositive profiles. Thus, the population of apoptotic cells following spinal cord contusion is comprised of oligodendrocytes and putative phagocytic microglia or macrophages. Given the delayed time course of oligodendrocyte death, the apoptotic death of oligodendrocytes may be amenable to pharmacological intervention with subsequent improvement in functional recovery.     

Morphometric study of myelinated fibers in human cervical spinal cord white matter

STUDY DESIGN: Using human autopsy spinal cord specimens, morphologic measurements of myelinated nerve fibers were performed, focusing on the regions that include the main white matter conduction paths. The hemilateral spinal cord morphology was also measured, and its relation with the component myelinated nerve fibers determined. OBJECTIVES: To determine the relation between spinal cord transverse area in the normal lower cervical spine, the site most vulnerable to chronic compressive myelopathy, and myelinated nerve fibers. SUMMARY OF BACKGROUND DATA: Considerable interindividual variation normally is observed in the morphology of the spinal cord transverse area. The influence of this variation on the composition of the white matter myelinated nerve fibers is obscure. METHODS: The C7 segments from seven cadavers were resected, and from magnified photographs of paraffin-embedded specimens, the hemilateral spinal cord area and funicular area were measured. Nerve fiber morphology was measured using Epon-embedded specimens. Three regions that included the main conduction paths were sampled, and magnified photographs obtained. The nerve fiber transverse morphology was measured using the ellipse conversion method, and the myelinated nerve density and fiber area were determined. RESULTS: Marked interindividual variations were found in both the hemilateral spinal cord transverse area and funicular area. A positive correlation was noted between the two, with the spinal cord transverse area large in the cases with a large funicular area. For fiber density and area, histograms were constructed that showed characteristic distribution patterns in each region. By dividing each region into two components (i.e., small- and large-diameter fibers), it was found that the interindividual variation in large-diameter fiber density was small, clarifying that the absolute number of large-diameter fibers compared to fiber density is more strongly dependent on the funicular area. CONCLUSIONS: The absolute number of large-diameter myelinated fibers is smaller in cross-sections of thin as compared to those of thick spinal cord. When elucidating the pathophysiology of compressive myelopathy, it is necessary to study not only the circumstances surrounding the spinal cord, but this kind of factor intrinsic to the spinal cord itself.     

Oligodendrocyte precursors originate from both the dorsal and the ventral parts of the spinal cord

Oligodendrocytes have been recently claimed to originate from bilateral columns of precursors whose extension is limited to the ventral region of the neuroepithelium. We designed an experiment in which the developmental capabilities of the different regions of the ventricular epithelium could be tested. We exchanged isotopically and isochronically defined sectors of the E2 spinal cord between quail and chick embryos and followed the production and migration of oligodendrocytes by using a quail-specific cDNA probe encoding the oligodendrocyte marker Schwann cell myelin protein. We showed that oligodendrocytes are generated in vivo from both ventral and dorsal halves of the neural tube. Moreover, extensive ventrodorsal, as well as dorsoventral, migrations of oligodendrogenic cells take place during spinal cord differentiation.     

Anoxic and ischemic injury of myelinated axons in CNS white matter: from mechanistic concepts to therapeutics

White matter of the brain and spinal cord is susceptible to anoxia and ischemia. Irreversible injury to this tissue can have serious consequences for the overall function of the CNS through disruption of signal transmission. Myelinated axons of the CNS are critically dependent on a continuous supply of energy largely generated through oxidative phosphorylation. Anoxia and ischemia cause rapid energy depletion, failure of the Na(+)-K(+)-ATPase, and accumulation of axoplasmic Na+ through noninactivating Na+ channels, with concentrations approaching 100 mmol/L after 60 minutes of anoxia. Coupled with severe K+ depletion that results in large membrane depolarization, high [Na+]i stimulates reverse Na(+)-Ca2+ exchange and axonal Ca2+ overload. A component of Ca2+ entry occurs directly through Na+ channels. The excessive accumulation of Ca2+ in turn activates various Ca(2+)-dependent enzymes, such as calpain, phospholipases, and protein kinase C, resulting in irreversible injury. The latter enzyme may be involved in "autoprotection," triggered by release of endogenous gamma-aminobutyric acid and adenosine, by modulation of certain elements responsible for deregulation of ion homeostasis. Glycolytic block, in contrast to anoxia alone, appears to preferentially mobilize internal Ca2+ stores; as control of internal Ca2+ pools is lost, excessive release from this compartment may itself contribute to axonal damage. Reoxygenation paradoxically accelerates injury in many axons, possibly as a result of severe mitochondrial Ca2+ overload leading to a secondary failure of respiration. Although glia are relatively resistant to anoxia, oligodendrocytes and the myelin sheath may be damaged by glutamate released by reverse Na(+)-glutamate transport. Use-dependent Na+ channel blockers, particularly charged compounds such as QX-314, are highly neuroprotective in vitro, but only agents that exist partially in a neutral form, such as mexiletine and tocainide, are effective after systemic administration, because charged species cannot penetrate the blood-brain barrier easily. These concepts may also apply to other white matter disorders, such as spinal cord injury or diffuse axonal injury in brain trauma. Moreover, whereas many events are unique to white matter injury, a number of steps are common to both gray and white matter anoxia and ischemia. Optimal protection of the CNS as a whole will therefore require combination therapy aimed at unique steps in gray and white matter regions, or intervention at common points in the injury cascades.     

Regeneration of lesioned corticospinal tract fibers in the adult rat spinal cord under experimental conditions

The absence of fiber regrowth in the injured spinal cord and brain is influenced by several different factors and mechanisms. Among these are factors which inhibit neurite growth which are found on the surface of oligodendrocytes and central myelin. Their neutralization by a specific antibody allowed regeneration of transected corticospinal tract fibers in the adult rat spinal cord. Using a recently introduced novel neuroanatomical tracer, biotin-dextran-amine, we demonstrate the extensive regenerative sprouting of lesioned corticospinal fibers in the lesioned adult spinal cord. In the presence of the antibody against the myelin-associated neurite growth inhibitors, some of these fibers grew over remaining tissue bridges into the caudal spinal cord. They branched extensively in the lumbar spinal cord segments. These branches were decorated with synapse-like boutons. This neuroanatomical configuration probably contributes importantly to the functional recovery observed earlier in these antibody-treated animals.     

Myelin formation by mouse glia in myelin-deficient rats treated with cyclosporine

Previous attempts to generate myelin in the myelin-deficient rat spinal cord by transplanting mouse glia were not successful. In order to determine whether this result was due to graft rejection or to interspecies mismatch of cellular or molecular components at the axoglial junction, we have repeated the experiment in cyclosporine-treated rats. Our results show that in the immunosuppressed hosts, foetal glial xenografts form an abundance of myelin within the dorsal columns at or near the injection site about two weeks after the operation. In some cases, myelination extends virtually across the entire width of the dorsal columns. Ultrastructurally, the myelin sheaths are normal in all respects, including the presence of the 'radial component'. The lateral edges of the myelin lamellae form typical paranodal axoglial junctions, some displaying periodic 'transverse bands'. We infer that previous mouse to rat xenograft failures reflect host immune response rather than mismatch of heterologous junctional components. We also compared foetal, early post-natal and adult xenografts. Foetal donor cells, containing an abundance of precursors but virtually no mature oligodendrocytes, are more effective than neonatal donor cells in forming myelin, and after adult grafts, we found no myelin formation. Thus, in xenografts, as in allografts, foetal precursor cells are far more suitable than glia from mature donors in generating significant amounts of myelin.     

Regeneration of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells

Precisely localized focal stereotaxic electrolytic lesions were made in the corticospinal tract at the level of the first to second cervical segments in the adult rat. This consistently destroyed all central nervous tissue elements (axons, astrocytes, oligodendrocytes, microglia, and microvessels) in a highly circumscribed area. In a group of these rats immediately after lesioning, a suspension of cultured adult olfactory ensheathing cells was transplanted into the lesion site. Within the first week after transplantation, the cut corticospinal axons (identified by anterograde transport of biotin dextran) extended caudally along the axis of the corticospinal tract as single, fine, minimally branched sprouts that ended in a simple tip, often preceded by a small varicosity. By 3 weeks, the regenerating axons, ensheathed by P0-positive peripheral myelin had accumulated into parallel bundles, which now extended across the full length of the lesioned area and reentered the caudal part of the host corticospinal tract. The transplants contained two main types of cells: (1) p75-expressing S cells, which later formed typical peripheral one-to-one myelin sheaths around individual ensheathed axons, and (2) fibronectin-expressing A cells, which aggregated into tubular sheaths enclosing bundles of myelinated axons. The point of reentry of the axons into the central nervous territory of the caudal host corticospinal tract was marked by the resumption of oligodendrocytic myelination. Thus the effect of the transplant was to form a "patch" of peripheral-type tissue across which the cut central axons regenerated and then continued to grow along their original central pathway.     

Functional analysis of the two-gene lysis system of the pneumococcal phage Cp-1 in homologous and heterologous host cells

Myelin-associated glycoprotein (MAG) was postulated to play an important role in myelination. However, we showed previously that MAG null mutants exhibited no gross abnormality in myelination. Ultrastructural studies revealed subtle alterations in periaxonal organisation, indicating a restricted structural role for MAG in the formation and maintenance of periaxonal structures (Li et al., 1994). Here we show that myelination in MAG deficient mice is not as finely controlled as it is in wild type mice. The abnormalities manifest themselves as a decrease in the proportion of myelinated axons and a reciprocal increase in the proportion of unmyelinated axons in mutants' optic nerves. In addition, dysregulated myelination is occasionally observed in the form of multiply myelinated fibres, grouping of myelinated axons and myelin debris by a large myelin sheath, redundant myelin loops and, very rarely, massive myelin surrounding relatively small axons. Thus, in the absence of MAG, some glial cells seem unable to determine when, where and how much myelin should be laid down. These data support the notion of MAG being a glial recognition/adhesion molecule. A model is proposed regarding the roles MAG could play in the formation and maintenance of myelin structure.     

Protection of the axonal cytoskeleton in anoxic optic nerve by decreased extracellular calcium

Since CNS white matter tracts contain axons, oligodendrocytes and astrocytes but not synapses, it is likely that anoxic injury of white matter is mediated by cellular mechanisms that do not involve synapses. In order to test the hypothesis, that anoxic injury of white matter is mediated by an influx of Ca2+ into the intracellular compartment of axons, we compared the ultrastructure of axons in rat optic nerve exposed to 60 min of anoxia in artificial cerebrospinal fluid (aCSF) containing normal (2 mM) Ca2+, and in aCSF containing zero-Ca2+ together with 5 mM EGTA. Optic nerves fixed at the end of 60 min of anoxia in 2 mM Ca2+ exhibit extensive ultrastructural alterations including disruption of microtubules and neurofilaments within the axonal cytoskeleton, development of membranous profiles and empty spaces between the axon and the ensheathing myelin, and swelling of mitochondria with loss of cristae. Bathing the nerves in zero-Ca2+ aCSF during anoxia protected the axons from cytoskeletal changes; after 60 min of anoxia, optic nerve axons retained normal-appearing microtubules and neurofilaments. Membranous profiles were rare, and empty spaces between axons and myelin did not develop in anoxic optic nerves bathed in zero-Ca2+ aCSF. Disorganization of cristae in axonal mitochondria was observed in anoxic optic nerves even when Ca2+ was omitted from the medium. Because Ca(2+)-mediated injury is known to disrupt the axonal cytoskeleton, these results support the hypothesis that anoxia triggers an abnormal influx of Ca2+ into myelinated axons in CNS white matter.