White matter involvement after TBI: Clues to axon and myelin repair capacity.

Impact-acceleration forces to the head cause traumatic brain injury (TBI) with damage in white matter tracts comprised of long axons traversing the brain. White matter injury after TBI involves both traumatic axonal injury (TAI) and myelin pathology that evolves throughout the post-injury time course. The axon response to initial mechanical forces and secondary insults follows the process of Wallerian degeneration, which initiates as a potentially reversible phase of intra-axonal damage and proceeds to an irreversible phase of axon fragmentation. Distal to sites of axon disconnection, myelin sheaths remain for prolonged periods, which may activate neuroinflammation and inhibit axon regeneration. In addition to TAI, TBI can cause demyelination of intact axons. These evolving features of axon and myelin pathology also represent opportunities for repair. In experimental TBI, demyelinated axons exhibit remyelination, which can serve to both protect axons and facilitate recovery of function. Myelin remodeling may also contribute to neuroplasticity. Efficient clearance of myelin debris is a potential target to attenuate the progression of chronic pathology. During the early phase of Wallerian degeneration, interventions that prevent the transition from reversible damage to axon disconnection warrant the highest priority, based on the poor regenerative capacity of axons in the CNS. Clinical evaluation of TBI will need to address the challenge of accurately detecting the extent and stage of axon damage. Distinguishing the complex white matter changes associated with axons and myelin is necessary for interpreting advanced neuroimaging approaches and for identifying a broader range of therapeutic opportunities to improve outcome after TBI.

Myelin and oligodendrocyte lineage cells in white matter pathology and plasticity after traumatic brain injury.

Impact to the head or rapid head acceleration-deceleration can cause traumatic brain injury (TBI) with a characteristic pathology of traumatic axonal injury (TAI) and secondary damage in white matter tracts. Myelin and oligodendrocyte lineage cells have significant roles in the progression of white matter pathology after TBI and in the potential for plasticity and subsequent recovery. The myelination pattern of specific brain regions, such as frontal cortex, may also increase susceptibility to neurodegeneration and psychiatric symptoms after TBI. White matter pathology after TBI depends on the extent and distribution of axon damage, microhemorrhages and/or neuroinflammation. TAI occurs in a pattern of damaged axons dispersed among intact axons in white matter tracts. TAI accompanied by bleeding and/or inflammation produces focal regions of overt tissue destruction, resulting in loss of both axons and myelin. White matter regions with TAI may also exhibit demyelination of intact axons. Demyelinated axons that remain viable have the potential for remyelination and recovery of function. Indeed, animal models of TBI have demonstrated demyelination that is associated with evidence of remyelination, including oligodendrocyte progenitor cell proliferation, generation of new oligodendrocytes, and formation of thinner myelin. Changes in neuronal activity that accompany TBI may also involve myelin remodeling, which modifies conduction efficiency along intact myelinated fibers. Thus, effective remyelination and myelin remodeling may be neurobiological substrates of plasticity in neuronal circuits that require long-distance communication. This perspective integrates findings from multiple contexts to propose a model of myelin and oligodendrocyte lineage cell relevance in white matter injury after TBI. This article is part of the Special Issue entitled 'Oligodendrocytes in Health and Disease'.

Components of myelin damage and repair in the progression of white matter pathology after mild traumatic brain injury.

White matter tracts are highly vulnerable to damage from impact-acceleration forces of traumatic brain injury (TBI). Mild TBI is characterized by a low density of traumatic axonal injury, whereas associated myelin pathology is relatively unexplored. We examined the progression of white matter pathology in mice after mild TBI with traumatic axonal injury localized in the corpus callosum. Adult mice received a closed-skull impact and were analyzed from 3 days to 6 weeks post-TBI/sham surgery. At all times post-TBI, electron microscopy revealed degenerating axons distributed among intact fibers in the corpus callosum. Intact axons exhibited significant demyelination at 3 days followed by evidence of remyelination at 1 week. Accordingly, bromodeoxyuridine pulse-chase labeling demonstrated the generation of new oligodendrocytes, identified by myelin proteolipid protein messenger RNA expression, at 3 days post-TBI. Overall oligodendrocyte populations, identified by immunohistochemical staining for CC1 and/or glutathione S-transferase pi, were similar between TBI and sham mice by 2 weeks. Excessively long myelin figures, similar to redundant myelin sheaths, were a significant feature at all post-TBI time points. At 6 weeks post-TBI, microglial activation and astrogliosis were localized to areas of axon and myelin pathology. These studies show that demyelination, remyelination, and excessive myelin are components of white matter degeneration and recovery in mild TBI with traumatic axonal injury.

Dysmyelination with preservation of transverse bands in a long-lived allele of the quaking mouse.

The new mutant mouse shaking (shk) differs from other "myelin mutants" in having a more stable neurological impairment and a much longer lifespan. We have shown that transverse bands (TBs), the component of the paranodal junction (PNJ) that attaches the myelin sheath to the axon, are present in the shk central nervous system (CNS), in contrast to more severely affected mutants, in which TBs are absent or rare. We have proposed that TBs are the major determinant underlying shk neurological stability and longevity. Here we report that TBs are abundant not only in the shk CNS but also in its peripheral nervous system (PNS), which, as in other "myelin mutants", is not as severely dysmyelinated as the CNS but does display structural abnormalities likely to affect impulse propagation. In particular, myelin sheaths are thinner than normal, and some axonal segments lack myelin sheaths entirely. In addition, we establish that the shk mutation, previously localized to chromosome 17, is a quaking (qk) allele consisting of a 105-nucleotide insertion in the qk regulatory region that decreases qk transcription but does not extend to the Parkin and Parkin coregulated genes, which are affected in the qk allele. We conclude that: 1) dysmyelination is less severe in the shk PNS than in the CNS, but TBs, which are present in both locations, stabilize the PNJs and prevent the progressive neurological deficits seen in mutants lacking TBs; and 2) the insertional mutation in shk mice is sufficient to produce the characteristic neurological phenotype without involvement of the Parkin and Parkin coregulated genes.

Comparison of cortical and white matter traumatic brain injury models reveals differential effects in the subventricular zone and divergent Sonic hedgehog signaling pathways in neuroblasts and oligodendrocyte progenitors.

The regenerative capacity of the central nervous system must be optimized to promote repair following traumatic brain injury (TBI) and may differ with the site and form of damage. Sonic hedgehog (Shh) maintains neural stem cells and promotes oligodendrogenesis. We examined whether Shh signaling contributes to neuroblast (doublecortin) or oligodendrocyte progenitor (neural/glial antigen 2 [NG2]) responses in two distinct TBI models. Shh-responsive cells were heritably labeled in vivo using Gli1-CreER(T2);R26-YFP bitransgenic mice with tamoxifen administration on Days 2 and 3 post-TBI. Injury to the cerebral cortex was produced with mild controlled cortical impact. Yellow fluorescent protein (YFP) cells decreased in cortical lesions. Total YFP cells increased in the subventricular zone (SVZ), indicating Shh pathway activation in SVZ cells, including doublecortin-labeled neuroblasts. The alternate TBI model produced traumatic axonal injury in the corpus callosum. YFP cells decreased within the SVZ and were rarely double labeled as NG2 progenitors. NG2 progenitors increased in the cortex, with a similar pattern in the corpus callosum. To further test the potential of NG2 progenitors to respond through Shh signaling, Smoothened agonist was microinjected into the corpus callosum to activate Shh signaling. YFP cells and NG2 progenitors increased in the SVZ but were not double labeled. This result indicates that either direct Smoothened activation in NG2 progenitors does not signal through Gli1 or that Smoothened agonist acts indirectly to increase NG2 progenitors. Therefore, in all conditions, neuroblasts exhibited differential Shh pathway utilization compared with oligodendrocyte progenitors. Notably, cortical versus white matter damage from TBI produced opposite responses of Shh-activated cells within the SVZ.

Oligodendrocyte lineage and subventricular zone response to traumatic axonal injury in the corpus callosum.

Traumatic brain injury frequently causes traumatic axonal injury (TAI) in white matter tracts. Experimental TAI in the corpus callosum of adult mice was used to examine the effects on oligodendrocyte lineage cells and myelin in conjunction with neuroimaging. The injury targeted the corpus callosum over the subventricular zone, a source of neural stem/progenitor cells. Traumatic axonal injury was produced in the rostral body of the corpus callosum by impact onto the skull at the bregma. During the first week after injury, magnetic resonance diffusion tensor imaging showed that axial diffusivity decreased in the corpus callosum and that corresponding regions exhibited significant axon damage accompanied by hypertrophic microglia and reactive astrocytes. Oligodendrocyte progenitor proliferation increased in the subventricular zone and corpus callosum. Oligodendrocytes in the corpus callosum shifted toward upregulation of myelin gene transcription. Plp/CreER(T):R26IAP reporter mice showed normal reporter labeling of myelin sheaths 0 to 2 days after injury but labeling was increased between 2 and 7 days after injury. Electron microscopy revealed axon degeneration, demyelination, and redundant myelin figures. These findings expand the cell types and responses to white matter injuries that inform diffusion tensor imaging evaluation and identify pivotal white matter changes after TAI that may affect axon vulnerability vs. recovery after brain injury.

Molecular architecture of myelinated nerve fibers: leaky paranodal junctions and paranodal dysmyelination.

Myelinated nerve fibers have evolved to optimize signal propagation. Each myelin segment is attached to the axon by the unique paranodal axoglial junction (PNJ), a highly complex structure that serves to define axonal ion channel domains and to direct nodal action currents through adjacent nodes. Surprisingly, this junction does not entirely seal the paranodal myelin sheath to the axon and thus does not entirely isolate the perinodal space from the internodal periaxonal space. Rather the paranode is penetrated by extracellular pathways between the myelin sheath and the axolemma for movement of molecules and the flow of current to and from the internodal axon. This review summarizes past and current studies demonstrating these pathways and considers what functional roles they subserve. In addition, modern genetic engineering methods permit modification of individual PNJ constituents, which provides an opportunity to define their specific functions. One component in particular, the transverse bands, plays a key role in maintaining the structure and function of the PNJ. Loss of transverse bands results not in frank demyelination but rather in subtle dysmyelination, which causes significant functional impairment. The consequences of such subtle defects in the PNJ are considered along with the relevance of these studies to human diseases of myelin.

FGF2 and FGFR1 signaling regulate functional recovery following cuprizone demyelination.

In demyelinating diseases, such as multiple sclerosis, remyelination offers the potential to recover function of viable denuded axons by restoring saltatory conduction and/or protecting from further damage. Mice with genetic reduction of fibroblast growth factor 2 (Fgf2) or Fgf receptor 1 (Fgfr1) exhibit dramatically improved remyelination following experimental demyelination with cuprizone. The current studies are the first to test neurobehavioral outcomes with these gene deletions that improved remyelination. The cuprizone protocols used did not produce overt abnormalities but did reduce bilateral sensorimotor coordination (complex wheel task) and increase sociability (two chamber apparatus with novel mouse). A significant effect of genotype was observed on the complex wheel task but not in the sociability apparatus. Specifically, complex wheel velocities for Fgf2 nulls improved significantly after removal of cuprizone from the diet. This improvement in Fgf2 null mice occurred following either acute (6 weeks) or chronic (12 weeks) demyelination. Plp/CreERT:Fgfr1(fl/fl) mice administered tamoxifen at 10 weeks of cuprizone treatment to induce Fgfr1 knockdown also showed improved recovery of running velocities on the complex wheels. Therefore, constitutive deletion of Fgf2 or Fgfr1 knockdown in oligodendrocyte lineage cells is sufficient to overcome impairment of sensorimotor coordination after cuprizone demyelination.

Paranodal permeability in "myelin mutants".

Fluorescent dextran tracers of varying sizes have been used to assess paranodal permeability in myelinated sciatic nerve fibers from control and three "myelin mutant" mice, Caspr-null, cst-null, and shaking. We demonstrate that in all of these the paranode is permeable to small tracers (3 kDa and 10 kDa), which penetrate most fibers, and to larger tracers (40 kDa and 70 kDa), which penetrate far fewer fibers and move shorter distances over longer periods of time. Despite gross diminution in transverse bands (TBs) in the Caspr-null and cst-null mice, the permeability of their paranodal junctions is equivalent to that in controls. Thus, deficiency of TBs in these mutants does not increase the permeability of their paranodal junctions to the dextrans we used, moving from the perinodal space through the paranode to the internodal periaxonal space. In addition, we show that the shaking mice, which have thinner myelin and shorter paranodes, show increased permeability to the same tracers despite the presence of TBs. We conclude that the extent of penetration of these tracers does not depend on the presence or absence of TBs but does depend on the length of the paranode and, in turn, on the length of "pathway 3," the helical extracellular pathway that passes through the paranode parallel to the lateral edge of the myelin sheath.

Permeability of the paranodal junction of myelinated nerve fibers.

We have used fluorescent dextran tracers to test the tightness of the paranodal junction of living or fixed myelinated fibers in mouse sciatic nerve. Both 3 and 70 kDa tracers are able to penetrate from the perinodal space symmetrically into the paranodes on either side of the node of Ranvier at a rate consistent with diffusion through an elongated helical pathway between the paranodal terminal loops of the myelin sheath. This pathway thus provides an access route for movement of water soluble nutrients and metabolites to and from the internodal axon and constitutes a pathway through which juxtaparanodal potassium channels may be activated and may in turn affect nodal excitability. This pathway may also allow access of antibodies and toxic molecules to the internodal axon in paraneoplastic syndromes and demyelinating diseases.

Role of transverse bands in maintaining paranodal structure and axolemmal domain organization in myelinated nerve fibers: effect on longevity in dysmyelinated mutant mice.

The consequences of dysmyelination are poorly understood and vary widely in severity. The shaking mouse, a quaking allele, is characterized by severe central nervous system (CNS) dysmyelination and demyelination, a conspicuous action tremor, and seizures in approximately 25% of animals, but with normal muscle strength and a normal lifespan. In this study we compare this mutant with other dysmyelinated mutants including the ceramide sulfotransferase deficient (CST-/-) mouse, which are more severely affected behaviorally, to determine what might underlie the differences between them with respect to behavior and longevity. Examination of the paranodal junctional region of CNS myelinated fibers shows that "transverse bands," a component of the junction, are present in nearly all shaking paranodes but in only a minority of CST-/- paranodes. The number of terminal loops that have transverse bands within a paranode and the number of transverse bands per unit length are only moderately reduced in the shaking mutant, compared with controls, but markedly reduced in CST-/- mice. Immunofluorescence studies also show that although the nodes of the shaking mutant are somewhat longer than normal, Na(+) and K(+) channels remain separated, distinguishing this mutant from CST-/- mice and others that lack transverse bands. We conclude that the essential difference between the shaking mutant and others more severely affected is the presence of transverse bands, which serve to stabilize paranodal structure over time as well as the organization of the axolemmal domains, and that differences in the prevalence of transverse bands underlie the marked differences in progressive neurological impairment and longevity among dysmyelinated mouse mutants.

Subtle myelin defects in PLP-null mice.

This study explores subtle defects in the myelin of proteolipid protein (PLP)-null mice that could potentially underlie the functional losses and axon damage known to occur in this mutant and in myelin diseases including multiple sclerosis. We have compared PLP-null central nervous system (CNS) myelin with normal myelin using ultrastructural methods designed to emphasize fine differences. In the PLP-null CNS, axons large enough to be myelinated often lack myelin entirely or are surrounded by abnormally thin sheaths. Short stretches of cytoplasm persist in many myelin lamellae. Most strikingly, compaction is incomplete in this mutant as shown by the widespread presence of patent interlamellar spaces of variable width that can be labeled with ferricyanide, acting as an aqueous extracellular tracer. In thinly myelinated fibers, interlamellar spaces are filled across the full width of the sheaths. In thick myelin sheaths, they appear filled irregularly but diffusely. These patent spaces constitute a spiral pathway through which ions and other extracellular agents may penetrate gradually, possibly contributing to the axon damage known to occur in this mutant, especially in thinly myelinated fibers, where the spiral path length is shortest and most consistently labeled. We show also that the "radial component" of myelin is distorted in the mutant ("diagonal component"), extending across the sheaths at 45 degrees instead of 90 degrees. These observations indicate a direct or indirect role for PLP in maintaining myelin compaction along the external surfaces of the lamellae and to a limited extent, along the cytoplasmic surfaces as well and also in maintaining the normal alignment of the radial component.