Myelin Clearance Research Articles

Intracellular phospholipase A2 group IVA and group VIA play important roles in Wallerian degeneration and axon regeneration after peripheral nerve injury

We provide evidence that two members of the intracellular phospholipase A(2) family, namely calcium-dependent group IVA (cPLA(2) GIVA) and calcium-independent group VIA (iPLA(2) GVIA) may play important roles in Wallerian degeneration in the mouse sciatic nerve. We assessed the roles of these PLA(2)s in cPLA(2) GIVA(-/-) mice, and mice treated with a selective inhibitor of iPLA(2) GVIA (FKGK11). Additionally, the effects of both these PLA(2)s were assessed by treating cPLA(2) GIVA(-/-) mice with the iPLA(2) inhibitor. Our data suggest that iPLA(2) GVIA may play more of a role in the early stages of myelin breakdown, while cPLA(2) GIVA may play a greater role in myelin clearance by macrophages. Our results also show that the delayed myelin clearance and Wallerian degeneration after sciatic nerve crush injury in mice lacking cPLA(2) and iPLA(2) activities is accompanied by a delay in axon regeneration, target re-innervation and functional recovery. These results indicate that the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) contribute significantly to various aspects of Wallerian degeneration in injured peripheral nerves, which is then essential for successful axon regeneration. This work has implications for injury responses and recovery after peripheral nerve injuries in humans, as well as for understanding the slow clearance of myelin after CNS injury and its potential consequences for axon regeneration.

The Membrane Attack Complex of the Complement System Is Essential for Rapid Wallerian Degeneration

The complement (C) system plays an important role in myelin breakdown during Wallerian degeneration (WD). The pathway and mechanism involved are, however, not clear. In a crush injury model of the sciatic nerve, we show that C6, necessary for the assembly of the membrane attack complex (MAC), is essential for rapid WD. At 3 d after injury, pronounced WD occurred in wild-type animals, whereas the axons and myelin of C6-deficient animals appeared intact. Macrophage recruitment and activation was inhibited in C6-deficient rats. However, 7 d after injury, the distal part of the C6-deficient nerves appeared degraded. As a consequence of a delayed WD, more myelin breakdown products were present than in wild-type nerves. Reconstitution of the C6-deficient animals with C6 restored the wild-type phenotype. Treatment with rhC1INH (recombinant human complement 1 inhibitor) blocked deposition of activated C-cleaved products after injury. These experiments demonstrate that the classical pathway of the complement system is activated after acute nerve trauma and that the entire complement cascade, including MAC deposition, is essential for rapid WD and efficient clearance of myelin after acute peripheral nerve trauma.

Protracted myelin clearance hinders central primary afferent regeneration following dorsal rhizotomy and delayed neurotrophin-3 treatment

Regeneration within or into the CNS is thwarted by glial inhibition at the site of a spinal cord injury and at the dorsal root entry zone (DREZ), respectively. At the DREZ, injured axons and their distal targets are separated by degenerating myelin and an astrocytic glia limitans. The different glial barriers to regeneration following dorsal rhizotomy are temporally and spatially distinct. The more peripheral astrocytic barrier develops first, and is surmountable by neurotrophin-3 (NT-3) treatment; the more central myelin-derived barrier, which prevents dorsal horn re-innervation by NT-3-treated axons, becomes significant only after the onset of myelin degeneration. Here we test the hypothesis that in the presence of NT-3, axonal regeneration is hindered by myelin degeneration products. To do so, we used the Long Evans Shaker (LES) rat, in which oligodendrocytes do not make CNS myelin, but do produce myelin-derived inhibitory proteins. We show that delaying NT-3 treatment for 1 week in normal (LE) rats, while allowing axonal penetration of the glia limitans and growth within degenerating myelin, results in misdirected regeneration with axons curling around presumptive degenerating myelin ovoids within the CNS compartment of the dorsal root. In contrast, delaying NT-3 treatment in LES rats resulted in straighter, centrally-directed regenerating axons. These results indicate that regeneration may be best optimized through a combination of neurotrophin treatment plus complete clearance of myelin debris.

T cells contribute to lysophosphatidylcholine‐induced macrophage activation and demyelination in the CNS

We have previously shown that intraspinal microinjection of lysophosphatidylcholine (LPC), a potent demyelinating agent, results in a rapid but brief influx of T cells (between 6 and 12 h). This is accompanied by a robust activation of macrophages/microglia that leads to demyelination by 48 h. In the present study, we examined whether this brief influx of T cells contributes to the activation of macrophages/microglia and demyelination by injecting LPC into the dorsal column white matter of athymic Nude mice that lack T cells. We show that there is a significant reduction in macrophage/microglial activation and myelin clearance after LPC injection in Nude mice as compared with wildtype controls. We also show that there is no difference in the recruitment of hematogenous macrophages into the spinal cord after LPC injection in the two mouse strains. Of the T cell cytokines assessed, there was a marked reduction in the mRNA expression of interleukin-2 (IL-2) in Nude mice compared with wildtype animals. Neutralizing IL-2 with function-blocking antibodies in wildtype animals resulted in a significant decrease in the number of phagocytic macrophages/microglia and a reduction in demyelination induced by LPC. While there may be other defects in Nude mice that might contribute to the effects shown here, these data suggest that the brief influx of T cells in this model of chemically-induced demyelination could play a role in macrophage/microglial activation and demyelination. These results may also have implications for remyelination in this and other types of CNS damage.

Exogenous Tissue Plasminogen Activator Enhances Peripheral Nerve Regeneration and Functional Recovery After Injury In Mice

Tissue plasminogen activator (tPA) is an essential component of the proteolytic cascade that lyses blood clots. Various studies also suggest that tPA plays important roles in the nervous system. We show that exogenous tPA or tPA/plasminogen (plg) promotes axonal regeneration, remyelination, and functional recovery after sciatic nerve injury in the mouse. Local application of tPA or tPA/plg 7 days after sciatic nerve crush significantly increased the total number of axons and myelinated axons, which is accompanied by enhanced expression of neurofilament. Treatment with tPA or tPA/plg reduced the deposition of fibrin(ogen) after nerve injury. Moreover, tPA or tPA/plg increased the number of macrophages and induced MMP-9 expression at the injury site, coincident with reduced collagen scar formation and accelerated clearance of myelin and lipid debris after treatment. Consequently, tPA or tPA/plg treatment protected muscles from atrophy after nerve injury, indicating better functional recovery. These results suggest that administration of exogenous tPA or tPA/plg promotes axonal regeneration and remyelination through removal of fibrin deposition and activation of MMP-9-positive macrophages, which may be responsible for myelin debris clearance and preventing collagen scar formation. Therefore, tPA may be useful for treatment of peripheral nerve injury.

Systemic injections of lipopolysaccharide accelerates myelin phagocytosis during Wallerian degeneration in the injured mouse spinal cord

The phagocytic cell response within the injured spinal cord is inefficient, allowing myelin debris to remain for prolonged periods of time within white matter tracts distal to the injury. Several proteins associated with this degenerating myelin are inhibitory to axon growth and therefore prevent severed axons from regenerating. Inflammatory agents such as lipopolysaccharide (LPS) can stimulate both the migration and phagocytic activity of macrophages. Using in situ hybridization, we found that the expression of the LPS membrane receptor, CD14, was enhanced in the mouse dorsal column following a dorsal hemisection. Double labeling studies showed that microglia and macrophages are the two major cell types expressing CD14 mRNA following spinal cord injury (SCI). We therefore tested whether systemic injections of LPS would increase the number and phagocytic activity of macrophages/microglia in the ascending sensory tract (AST) of the mouse dorsal column following a dorsal hemisection. Mice were treated daily via intraperitoneal injections of either LPS or phosphate-buffered saline (PBS). At 7 days post-SCI, greater numbers of activated mononuclear phagocytes were present in the AST undergoing Wallerian degeneration (WD) in LPS-treated animals compared with controls. Animals treated with LPS also exhibited greater Oil Red O staining, which is specific for degenerating myelin and macrophages phagocytosing myelin debris. Myelin clearance was confirmed at 7 days using Luxol Fast Blue staining and on toluidine blue-stained semi-thin sections. These results indicate that it is possible to manipulate the innate immune response to accelerate myelin clearance during WD in the injured mouse spinal cord.

Involvement of monocyte chemoattractant protein-1, macrophage inflammatory protein-1 and interleukin-1 in Wallerian degeneration

Wallerian degeneration in the CNS and PNS consists of degradation and phagocytosis of axons and their myelin sheath distal to the site of injury. This process of degeneration, which requires an effective macrophage response, occurs rapidly in peripheral nerves but is slow in the CNS. Rapid Wallerian degeneration in peripheral nerves may contribute to subsequent axonal regeneration. We show that there is a marked increase in mRNA expression of three pro-inflammatory molecules, the chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha), and the cytokine interleukin-1beta (IL-1beta), in the mouse sciatic nerve but not in the spinal cord undergoing Wallerian degeneration. Neutralizing MCP-1, MIP-1alpha and IL-1beta in the lesioned sciatic nerve with function-blocking antibodies suppressed macrophage responses and myelin clearance. Injecting recombinant MCP-1, MIP-1alpha or IL-1beta into the normal, uninjured spinal cord triggered the expression of a number of chemokines and cytokines. Furthermore, injecting recombinant MCP-1/MIP-1alpha or IL-1beta into the dorsal column of the spinal cord undergoing Wallerian degeneration triggered rapid macrophage/microglial activation and myelin clearance. These findings provide direct evidence that MCP-1, MIP-1alpha and IL-1beta are important regulators of macrophage responses that lead to rapid myelin breakdown and clearance in Wallerian degeneration.

Failed Central Nervous System Regeneration: A Downside of Immune Privilege?

Immunity is required to eliminate dangerous or degenerated material and to support regeneration, but also causes significant parenchymal damage. In the eye and the brain, in which cornea and lens poorly regenerate and neurons are hardly replaceable, early transplantation experiments demonstrated remarkable tolerance to various grafts. This "immunologically privileged status" (Billingham and Boswell, 1953) may reflect evolutionary pressure to downmodulate certain actions of immune cells within particularly vulnerable tissues. As an example, tolerating certain "neurotrophic" viruses may often be a more successful strategy for survival than the elimination of all infected neurons. While several constitutive and inducible signals maintaining or re-establishing immune tolerance within the brain have been identified, it has also become evident that the resulting anti-inflammatory environment limits certain beneficial effects of neuroinflammation such as neurotrophin secretion or glutamate buffering by T-cells and the clearance of growth-inhibiting myelin or amyloid. Following spinal cord injury, the costs and benefits of neuroinflammation seem to come close because enhancing as well as suppressing innate or adaptive immunity caused amelioration and aggravation of functional regeneration in similar experiments. Evaluating such balances has also begun in (animal models of) Alzheimer's disease, central nervous system trauma, and stroke, and the appreciation of the beneficial side of neuroinflammation has caused a rethinking of the ill-defined use of immune suppressants. As dual roles for individual molecules have been recognized (Merrill and Benveniste, 1996), we are uncovering an already fine-tuned system, but the challenge remains to further support beneficial immune cascades without causing additional damage, and vice versa.

Complement factors in adult peripheral nerve: a potential role in energy metabolism

Complement cascade factors are known to play a critical role in myelin clearance after peripheral nerve injury. Here we show that components of both the classical ( C1qa, C1qb, C1qc, C2 and C4) and alternative ( C3, B and adipsin) pathways are expressed by uninjured peripheral nerve as well. mRNAs of components of the alternative pathway were predominantly found in the peri/epineurium, although factor C3 and factor B were also detected in the endoneurial compartment of adult nerve. Interestingly, adipsin mRNA was detected only in peri/epineurium, while adipsin protein was present in both peri/epineurium and endoneurium. This suggests that adipsin is transported to the endoneurium via the circulation from the peri/epineurium or outside of the nerve. Factor 5 and factor 9, necessary for the formation of the membrane–attack complex, were not detected in any part of the healthy peripheral nerve, which together with the observed presence of negative regulators of complement activation, is likely to prevent damage to the healthy nerve caused by complement activation. By analogy with the known role of complement factors in fat, we propose that local expression of these factors plays a role in the regulation of fatty acid homeostasis in the nerve and, thereby, in energy metabolism cross-talk between different compartments of the peripheral nerve.

Expression of complement components in the peripheral nervous system.

We have generated a SAGE (serial analysis of gene expression) library of normal sciatic nerve and found tags encoding for mRNAs of the complement system highly represented. RNA (RT-PCR and northern blot hybridization) and protein (western blot analysis and immunohistochemistry) studies confirmed these findings. High expression of classical pathway components, alternative pathway components and inhibitory components was observed in specific regions of the sciatic nerve. The first components of complement were found in axons, whereas the inhibitory components were detected in the perineurium, thereby protecting the nerve from a complement attack. Immunoreactivity towards activated complement factors was noted in post traumatic neuromas and after acute crush injury, which exemplify nerve regeneration and degeneration. We propose that local production of complement in the peripheral nervous system participates in the protection of healthy nerve and is needed for efficient clearance of myelin after injury: a prerequisite for normal regeneration and remyelination of the peripheral nerve.

Nogo-C is sufficient to delay nerve regeneration

Axonal regeneration succeeds in the peripheral but not central nervous system of adult mammals. Peripheral clearance of myelin coupled with selective CNS expression of axon growth inhibitors, such as Nogo, may account for this reparative disparity. To assess the sufficiency of Nogo for limiting axonal regeneration, we generated transgenic mice expressing Nogo-C in peripheral Schwann cells. Nogo-C includes the panisoform inhibitory Nogo-66 domain, but not a second Nogo-A-specific inhibitory domain, allowing a selective consideration of the Nogo-66 region. The oct-6::nogo-c transgenic mice regenerate axons less rapidly than do wild-type mice after mid-thigh sciatic nerve crush. The delayed axonal regeneration is associated with a decreased recovery rate for motor function after sciatic nerve injury. Thus, expression of the Nogo-66 domain by otherwise permissive myelinating cells is sufficient to hinder axonal reextension after trauma.

Myelin phagocytosis by macrophages and nonmacrophages during Wallerian degeneration.

The literature concerning Schwann cells (SCs) and macrophages in myelin phagocytosis during Wallerian degeneration is reviewed. SCs carry out the first step in the removal of myelin by segmenting myelin and then incorporating the degraded myelin. The recruited macrophages then join in the myelin-phagocytosis event, appearing to make full use of their original phagocyte abilities until the end of myelin clearance. The molecular mechanisms of the two cells underlying myelin phagocytosis are thought to be different; myelin phagocytosis by SCs being lectin-mediated, i.e., opsonin-independent, whereas that of macrophages is mainly opsonin-dependent. It is important to note that SCs and macrophages cooperatively accomplish myelin phagocytosis.

Myelin debris clearance during Wallerian degeneration in the goldfish visual system

We have examined the clearance of myelin debris from the visual pathways of the goldfish during Wallerian degeneration. Both the rate and pattern of myelin disappearance from the optic nerve and tract were determined using immunohistochemistry on frozen sections, as well as plastic sections and electron microscopy. Animals with and without regenerating optic axons were examined in order to determine whether the axons play a role in myelin clearance. We found that myelin is cleared at different rates along the visual paths. Thus, virtually all myelin debris is gone in the optic tract and distal optic nerve stump by 4 weeks after surgery, while in the cranial nerve segment, myelin clearance is still incomplete at 6 weeks postoperative. These temporal and spatial patterns of myelin clearance are the same in animals with and without regenerating axons, thus indicating that growing axons do not influence this process. Finally, ultrastructural observations revealed that both astrocytes and microglia participate in phagocytosing myelin debris in the optic nerve, while in the tract, the vast majority of debris is removed by microglia alone. These data are discussed with regard to possible mechanisms controlling the differential expression of myelin clearance.

Myelin debris clearance during Wallerian degeneration in the goldfish visual system

We have examined the clearance of myelin debris from the visual pathways of the goldfish during Wallerian degeneration. Both the rate and pattern of myelin disappearance from the optic nerve and tract were determined using immunohistochemistry on frozen sections, as well as plastic sections and electron microscopy. Animals with and without regenerating optic axons were examined in order to determine whether the axons play a role in myelin clearance. We found that myelin is cleared at different rates along the visual paths. Thus, virtually all myelin debris is gone in the optic tract and distal optic nerve stump by 4 weeks after surgery, while in the cranial nerve segment, myelin clearance is still incomplete at 6 weeks postoperative. These temporal and spatial patterns of myelin clearance are the same in animals with and without regenerating axons, thus indicating that growing axons do not influence this process. Finally, ultrastructural observations revealed that both astrocytes and microglia participate in phagocytosing myelin debris in the optic nerve, while in the tract, the vast majority of debris is removed by microglia alone. These data are discussed with regard to possible mechanisms controlling the differential expression of myelin clearance. J. Neurosci. Res. 59:47–62, 2000 © 2000 Wiley-Liss, Inc.

Differential response of macrophage subpopulations to myelin degradation in the injured rat sciatic nerve.

Molecular mechanisms of myelin removal by macrophages were explored by examining the immunophenotypes of macrophages following injury of rat sciatic nerve, using a combined method of immunohistochemistry and confocal laser microscopy. In the crush injury model, the involvement in myelin clearance of a cytoplasmic antigen specific for monocytes/macrophages, ED1, was evident. The obvious recruitment of ED1-immunoreactive (-ir) cells was detected first at the crush injury site and then in the distal stump within which Wallerian degeneration had occurred. Double labelling revealed that the ED1-ir cells, except for monocyte-like round cells, always phagocytosed myelin basic protein-ir myelin debris. On the other hand, the expression of ED2, a surface antigen specific for resident macrophages, was significantly different; ED2-ir cells also increased while myelin removal was progressing from day 3 to day 7, but only some of the cells were engaged in myelin phagocytosis. The poor capacity of myelin phagocytosis by ED2-ir cells was supported by the transection model, in which the proximal stump was ligated to suppress regeneration. ED2 may be involved in events other than myelin removal, providing a local environment conducive to axonal regeneration. Our findings thus seem to suggest that ED1 is one of the most reliable markers for cells carrying out myelin phagocytosis, whereas ED2 may participate in entirely different functions. The expression of complement receptor type 3, OX42, was similar to that of ED1 in terms of the swift recruitment of immunopositive cells, their distribution with close association to myelin debris and their high phagocytotic capacity. This supports previously reported in vitro evidence that myelin phagocytosis by macrophages may be complement-mediated.

Peripheral nerve-stimulated macrophages simulate a peripheral nerve-like regenerative response in rat transected optic nerve

We have previously demonstrated that the failure of the mammalian central nervous system (CNS) to regenerate following axonal injury is related to its immunosuppressive nature, which restricts the ability of both recruited blood-borne monocytes and CNS-resident microglia to support a process of repair. In this study we show that transected optic nerve transplanted with macrophages stimulated by spontaneously regenerating nerve tissue, e.g., segments of peripheral nerve (sciatic nerve), exhibit axonal regrowth at least as far as the optic chiasma. Axonal regrowth was confirmed by double retrograde labeling of the injured optic axons, visualized in their cell bodies. Transplanted macrophages exposed to segments of CNS (optic) nerve were significantly less effective in inducing regrowth. Immunocytochemical analysis showed that the induced regrowth was correlated with a wide distribution of macrophages within the transplanted-transected nerves. It was also correlated with an enhanced clearance of myelin, known to be inhibitory for regrowth and poorly eliminated after injury in the CNS. These results suggest that healing of the injured mammalian CNS, like healing of any other injured tissue, requires the partnership of the immune system, which is normally restricted, but that the restriction can be circumvented by transplantation of peripheral nerve-stimulated macrophages.

Complement Depletion Reduces Macrophage Infiltration and Activation during Wallerian Degeneration and Axonal Regeneration

After peripheral nerve injury, macrophages infiltrate the degenerating nerve and participate in the removal of myelin and axonal debris, in Schwann cell proliferation, and in axonal regeneration. In vitro studies have demonstrated the role serum complement plays in both macrophage invasion and activation during Wallerian degeneration of peripheral nerve. To determine its role in vivo, we depleted serum complement for 1 week in adult Lewis rats, using intravenously administered cobra venom factor. At 1 d after complement depletion the right sciatic nerve was crushed, and the animals were sacrificed 4 and 7 d later. Macrophage identification with ED-1 and CD11a monoclonal antibodies revealed a significant reduction in their recruitment into distal degenerating nerve in complement-depleted animals. Complement depletion also decreased macrophage activation, as indicated by their failure to become large and multivacuolated and their reduced capacity to clear myelin, which was evident at both light and electron microscopic levels. Axonal regeneration was delayed in complement-depleted animals. These findings support a role for serum complement in both the recruitment and activation of macrophages during peripheral nerve degeneration as well as a role for macrophages in promoting axonal regeneration.

Deficient activation of microglia during optic nerve degeneration

Transection of an optic nerve (ON) is followed by slow removal of myelin. We studied microglia for the expression of molecules that characterize activated myelin phagocytosing macrophages: MAC-1, FcγII/III receptor (FcR), MAC-2, and F4/80. In-vitro, microglia expressed all molecules and phagocytosed myelin. In-vivo, intact ON displayed high levels of MAC-1, little FcR and F4/80, and no MAC-2. The expression of these molecules was upregulated differentially in in-vivo degenerating ON: MAC-1 uniformly, FcR and F4/80 variably, and MAC-2 sporadically. The distribution of MAC-2 expression correlated best with a pattern of sporadic structural degeneration. Thus in-vivo, ON injury is followed by deficient microglia activation, which we suggest contributes significantly to the slow clearance of myelin.

Increased vulnerability to demyelination in streptozotocin diabetic rats.

Demyelination is a prominent feature in nerve biopsies of patients with diabetic neuropathy. The mechanism is unknown because diabetic rodents, unlike humans, do not consistently develop segmental demyelination. We examined how diabetes influences toxicant-induced demyelination, remyelination, Schwann cell nerve growth factor receptor (p75) expression, and endoneurial macrophage apolipoprotein E (apo E) synthesis in diabetic rats. Postnatal day 17 (P17) rats were given 110 mg/kg streptozotocin intraperitoneally and then fed a diet containing metallic tellurium (Te) from P20 to P27 to induce demyelination. Transverse electron micrographs and immunostained longitudinal cryosections were prepared from sciatic nerve during demyelination and remyelination. Diabetic rats had a mean serum glucose concentration of 490 mg/dl and consumed equivalent doses of peroral Te. The number of demyelinated fibers in electron micrographs was increased significantly by 17% (P < .0011). Endoneurial density of p75-stained Schwann cells was increased in diabetic rats in proportion to the increased number of injured internodes. Density of apo E- and ED1-positive macrophages also was significantly increased in diabetes. There was no delay in macrophage myelin clearance. and remyelination was not compromised. Increased Schwann cell vulnerability to stress, by increasing the turnover rate of myelinated units, may explain why myelin defects accumulate after long-standing diabetes.

Increased vulnerability to demyelination in streptozotocin diabetic rats

Demyelination is a prominent feature in nerve biopsies of patients with diabetic neuropathy. The mechanism is unknown because diabetic rodents, unlike humans, do not consistently develop segmental demyelination. We examined how diabetes influences toxicant-induced demyelination, remyelination, Schwann cell nerve growth factor receptor (p75) expression, and endoneurial macrophage apolipoprotein E (apo E) synthesis in diabetic rats. Postnatal day 17 (P17) rats were given 110 mg/kg streptozotocin intraperitoneally and then fed a diet containing metallic tellurium (Te) from P20 to P27 to induce demyelination. Transverse electron micrographs and immunostained longitudinal cryosections were prepared from sciatic nerve during demyelination and remyelination. Diabetic rats had a mean serum glucose concentration of 490 mg/dl and consumed equivalent doses of peroral Te. The number of demyelinated fibers in electron micrographs was increased significantly by 17% (P < .0011). Endoneurial density of p75-stained Schwann cells was increased in diabetic rats in proportion to the increased number of injured internodes. Density of apo E- and ED1-positive macrophages also was significantly increased in diabetes. There was no delay in macrophage myelin clearance, and remyelination was not compromised. Increased Schwann cell vulnerability to stress, by increasing the turnover rate of myelinated units, may explain why myelin defects accumulate after long-standing diabetes. © 1996 Wiley-Liss, Inc.