Myelin phagocytosis in Wallerian degeneration

Summary Myelin phagocytosis in Wallerian degeneration was studied using a model of murine sciatic nerve degeneration in millipore diffusion chambers in the peritoneal cavity of host mice. Immunocytological investigations showed the dependence of myelin digestion on the invasion of Fc-positive, Mac-1-positive and partly Ia-positive monocytes. Lymphocytes did not play a prominent role. Compared to Wallerian degeneration in situ, phagocytosis was decreased in nerves enclosed by millipore membranes on both sides of the chamber. The membrane acted as a trap for invading monocytes/macrophages. Neither tissue integrity nor genetic strain influenced the degree of phagocytosis. A modification of the experimental technique is introduced which permits myelin phagocytosis in the peritoneal cavity in a degree comparable to that in Wallerian degeneration in situ.Supported by a grant from the Deutsche Forschungsgemeinschaft (609)     

Ascorbic acid accelerates Wallerian degeneration after peripheral nerve injury

Wallerian degeneration occurs after peripheral nerve injury and provides a beneficial microenvironment for nerve regeneration. Our previous study demonstrated that ascorbic acid promotes peripheral nerve regeneration, possibly through promoting Schwann cell proliferation and phagocytosis and enhancing macrophage proliferation, migration, and phagocytosis. Because Schwann cells and macrophages are the main cells involved in Wallerian degeneration, we speculated that ascorbic acid may accelerate this degenerative process. To test this hypothesis, 400 mg/kg ascorbic acid was administered intragastrically immediately after sciatic nerve transection, and 200 mg/kg ascorbic acid was then administered intragastrically every day. In addition, rat sciatic nerve explants were treated with 200 μM ascorbic acid. Ascorbic acid significantly accelerated the degradation of myelin basic protein-positive myelin and neurofilament 200-positive axons in both the transected nerves and nerve explants. Furthermore, ascorbic acid inhibited myelin-associated glycoprotein expression, increased c-Jun expression in Schwann cells, and increased both the number of macrophages and the amount of myelin fragments in the macrophages. These findings suggest that ascorbic acid accelerates Wallerian degeneration by accelerating the degeneration of axons and myelin in the injured nerve, promoting the dedifferentiation of Schwann cells, and enhancing macrophage recruitment and phagocytosis. The study was approved by the Southern Medical University Animal Care and Use Committee(approval No. SMU-L2015081) on October 15, 2015.     

Nature of signals that initiate the immune response during Wallerian degeneration of peripheral nerves

Monocyte chemoattractant protein-1 is produced by Schwann cells during Wallerian degeneration of a peripheral nerve and contributes to a selective accumulation of macrophages in the degenerating segment. An in vitro preparation has been developed to analyze the molecules from axons and non-neuronal cells in nerves that stimulate an increased production of monocyte chemoattractant protein-1 mRNA by Schwann cells. For this purpose, Schwann cells obtained from neonatal rats were maintained in culture, exposed to putative molecular stimuli and analyzed for their content of monocyte chemoattractant protein-1 mRNA. Under basal conditions, the concentration of monocyte chemoattractant protein-1 in Schwann cells was low. Freeze-killed fragments or homogenates of nerve (or brain) but not viable nerve or freeze-killed muscle were effective in inducing monocyte chemoattractant protein-1 mRNA. The inductive activity was abolished by heating. Results of dialysis of supernatants of nerve homogenates indicate that a protein or proteins of 1-10 kDa were capable of stimulating synthesis of monocyte chemoattractant protein-1 by Schwann cells. Also, the activity in nerve homogenates was partially inhibited by antibodies to Toll-like receptor-4. The observations suggest that a non-secreted protein is released from disintegrating axons to initiate the innate immune response that characterizes Wallerian degeneration.     

Critical signaling pathways during Wallerian degeneration of peripheral nerve

Wallerian degeneration is a critical biological process that occurs in distal nerve stumps after nerve injury. To systematically investigate molecular changes underlying Wallerian degeneration, we used a rat sciatic nerve transection model to examine microarray analysis out-comes and investigate significantly involved Kyoto Enrichment of Genes and Genomes (KEGG) pathways in injured distal nerve stumps at 0, 0.5, 1, 6, 12, and 24 hours, 4 days, 1, 2, 3, and 4 weeks after peripheral nerve injury. Bioinformatic analysis showed that only one KEGG pathway (cytokine-cytokine receptor interaction) was significantly enriched at an early time point (1 hour post-sciatic nerve transection). At later time points, the number of enriched KEGG pathways initially increased and then decreased. Three KEGG pathways were studied in further detail: cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, and axon guidance. Moreover, temporal expression patterns of representative differentially expressed genes in these KEGG pathways were validated by real time-polymerase chain reaction. Taken together, the above three signaling pathways are important after sciatic nerve injury, and may increase our understanding of the molecular mechanisms underlying Wallerian degeneration.     

On the occurrence of the fenestrated vessels in Wallerian degeneration of the peripheral nerve

Summary Ultrastructural studies were made on the distal segments of the mouse phrenic nerve after crush injury. In the control, endoneurium contained only unfenestrated capillaries. In the experiment, from day 2 to day 6, endoneurial capillaries occasionally showed fenestrations with the attenuation of its cytoplasm. At this stage, axonal degeneration and myelin breakdown became evident showing early stage of Wallerian degeneration. In addition, detachment of the neighboring endothelial cells concomitant with the invasion of macrophage was also observed. These findings were previously unobserved changes of the endoneurial endothelium in Wallerian degeneration. The significance of the early occurrence of fenestrae was discussed briefly.     

Demyelination can proceed independently of axonal degradation during Wallerian degeneration in wlds mice

Peripheral nerve injury induces axonal degeneration and demyelination, which are collectively referred to as Wallerian degeneration. It is generally assumed that axonal degeneration is a trigger for the subsequent demyelination processes such as myelin destruction and de-differentiation of Schwann cells, but the detailed sequence of events that occurs during this initial phase of demyelination following axonal degeneration remains unclear. Here we performed a morphological analysis of injured sciatic nerves of wlds mice, a naturally occurring mutant mouse in which Wallerian degeneration shows a significant delay. The slow Wallerian degerenation phenotype of the wlds mutant mice would enable us to dissect the events that take place during the initial phase of demyelination. Ultrastrucural analysis using electron microscopy showed that the initial process of myelin destruction was activated in injured nerves of wlds mice even though they exhibit morphologically complete protection of axons against nerve injury. We also found that some intact axons were completely demyelinated in degenerating nerves of wlds mice. Furthermore, we observed that de-differentiation of myelinating Schwann cells gradually proceeded even though the axons remained morphologically intact. These data suggest that initiation and progression of demyelination in injured peripheral nerves is, at least in part, independent of axonal degeneration.     

Changes of myelin proteins during Wallerian degeneration in situ and in millipore diffusion chambers preventing active phagocytosis

Changes of myelin proteins in mouse sciatic nerves were studied comparing nerves degenerating in situ with nerves enclosed in millipore diffusion chambers which eliminate invasion of non-resident cells. Nerves kept in chambers showed nearly complete preservation of myelin sheaths with a very slow degradation of myelin proteins. Nerves degenerating in situ showed rapid myelin phagocytosis by macrophages with almost complete disappearance of myelin proteins after 28 days. These data elucidate the role of macrophages for removal of myelin proteins.     

Origin of macrophages in traumatic lesions and Wallerian degeneration in peripheral nerves

Summary The origin of the macrophages in peripheral nerves was investigated in newborn rabbits after labelling the blood monocytes with intravenous carbon suspensions.Carbon labelled lipid macrophages were observed at the site of trauma in the nerves but not in areas of Wallerian degeneration. It is suggested that in Wallerian degeneration, resorption of degenerate tissue is performed by endogenous cells only, while in traumatic lesions part of the phagocytosis is performed by migrated monocytes. The early increase in vascular permeability in traumatic lesions may be a prerequisite for the attraction of the monocytes.     

Liposome-mediated monocyte depletion during Wallerian degeneration defines the role of hematogenous phagocytes in myelin removal

Newly recruited hematogenous mononuclear cells of the monocyte/macrophage system are suggested to be important effector cells in myelin removal during Wallerian degeneration. Their role has extensively been studied in various in vitro and in vivo models. However, there has been much controversy concerning the role of hematogenous vs. resident cells of the peripheral nervous system in Wallerian degeneration. The present study used a recently established technique to deplete the hematogenous monocyte population by application of dichloromethylene diphosphonate-containing liposomes. Intravenously injected liposomes containing dichloromethylene diphosphonate (Cl₂MDP) are ingested by macrophages and monocytes and cause temporary and selective depletion of these cells. The number of LFA-1-and Mac-l- positive macrophages within the nerves was significantly reduced when liposomes were injected shortly after nerve transsection. In these nerves, myelin degradation was significantly less, indicating an essential role of newly recruited phagocytes in this process. Macrophage invasion of degenerating nerves occurred within the first 2 days after transsection. Resident cells of the peripheral nerve participate in myelin removal since macrophage depletion did not completely abolish myelin degradation. These results confirm the important role of hematogenous phagocytes in myelin removal during Wallerian degeneration. © 1996 Wiley-Liss, Inc.     

Differential expression and potential role of SOCS1 and SOCS3 in Wallerian degeneration in injured peripheral nerve

Pro-inflammatory chemokines and cytokines play an important role in Wallerian degeneration (WD) after peripheral nerve injury. These pro-inflammatory signals are “turned-off” in a timely manner to ensure that the inflammatory response in the injured nerve is limited. The factors that regulate the turning-off of the pro-inflammatory state are not fully understood. The suppressors of cytokine signaling (SOCS) proteins are potential candidates that could limit the inflammatory response by acting to regulate cytokine signaling at the intracellular level. In this work we show that the expression SOCS1 and SOCS3 proteins differ from each other during WD in the mouse sciatic nerve after cut/ligation and crush injuries. SOCS1 is mainly expressed by macrophages and its expression is inversely correlated with phosphorylation of JAK2 and STAT3 signaling proteins and the expression of pro-inflammatory cytokines IL-1β and TNFα. In addition, treatment of cut/ligated nerves, which express lower levels of SOCS1 as compared to crush injury, with a SOCS1 mimetic peptide leads to a decrease in macrophage numbers at 14 days post-injury and reduces IL-1β mRNA expression 1 day post-injury. In contrast, SOCS3 expression is restricted mainly to Schwann cells and is negatively correlated with the expression of IL-6 and LIF. These data suggest that SOCS1 and SOCS3 may play different roles in WD and provide a better understanding of some of the potential regulatory mechanisms that may control inflammation and regeneration in the injured peripheral nerve.     

Plasminogen activators in rat neural tissues during development and in Wallerian degeneration

Summary The fibrinolytic activity of blood is caused by plasminogen activators (PA) converting plasminogen to plasmin, the active fibrinolytic protease. PA activity in rat neural tissues was studied by Todd's fibrin slide technique. Cryostat sections overlayed with a film of plasminogen and fibrin were incubated for 60–90 min. PA activity was related to the size of the zone of fibrinolysis surrounding the sections. No lysis occurred with fibrin alone. In rats perfused with saline prior to decapitation the size of the zone of lysis was approximately the same as in non-perfused animals. PA activity was compared in the following tissues: adult (2–3 month) cerebellum and 6–14-day postnatal cerebellum; normal sciatic nerve and transected sciatic nerve 1–9 weeks after operation (in these experiments the sciatic nerve was crushed on the left side, on the right side it was transected and the stumps were tightly ligated to prevent regeneration); normal optic nerves and optic nerves undergoing Wallerian degeneration 1–2 weeks after enucleation of the eye. As compared to normal cerebellum PA activity was increased in 6–14-day cerebellum. PA activity was also markedly increased in both crushed and ligated sciatic nerves 1–4 weeks after operation while no differences were observed between normal sciatic nerves and sciatic nerves 9 weeks after ligation. The zone of fibrinolysis surrounding normal optic nerves and the optic nerves of blinded rats was approximately the same. It is proposed that the fibrinolytic system may be relevant to the problem of CNS regeneration.Supported by the Veterans Administration     

The role of Schmidt-Lanterman incisures in Wallerian degeneration

Summary The number of the Schmidt-Lanterman incisures and their intrasegmental distribution were studied at 36 h after transection of the rat sciatic nerve. Examination of teased, proximo-distally oriented, myelinated nerve fibers revealed no difference between the distal and the proximal stump. The results indicate that no proliferation of the incisures is required for the fiber fragmentation: numerous incisures are normally available in the midinternodal area where the degeneration begins.     

Inhibitory effects of thalidomide on cellular proliferation,endoneurial edema and myelin phagocytosis during early Wallerian degeneration

In addition to the well-known teratogenic effect of thalidomide, previous studies have revealed mild immunosuppressive properties and, more recently, and antiangiogenic activity. To find out more about the specificity of these effects we studied the influence of orally administered thalidomide on Wallerian degeneration in rats. Wallerian degeneration is a potent experimental model for studying reproducible cell proliferation in vivo. Examination of distal nerve segments of transected sciatic nerves from rats that had been treated with thalidomide (2×250 mg/kg per day) revealed a significant reduction of endoneurial cell counts at 10–15 days after surgery compared to that seen in controls. This effect was not statistically significant, at a very early stage of Wallerian degeneration, i.e., at 5 days after transection of the nerve. Subperineurial edema and phagocytosis was also reduced, although this was not statistically significant. This apparently nonspecific inhibitory effect of thalidomide during early Wallerian degeneration shown in the present study should be investigated further for its possible relationship to other previously established inhibitory activities of thalidomide, especially its immunosuppressive effect in man.The results of this study were presented in part at the First Meeting of the Peripheral Nerve Society, 12-16 June 1994, in St. Paul, Minn., USA, and at the 39th Annual Meeting of the Deutsche Gesellschaft für Neuropathologie and Neuroanatomie, 5–8 October 1994, in München, Germany [15]     

Induction of neuropilins-1 and -2 and their ligands, Sema3A, Sema3F, and VEGF, during Wallerian degeneration in the peripheral nervous system

The neuropilins, NP-1 and NP-2, are coreceptors for Sema3A and Sema3F, respectively, both of which are repulsive axonal guidance molecules. NP-1 and NP-2 are also coreceptors for vascular endothelial growth factor (VEGF). The neuropilins and their ligands are known to play prominent roles in axonal pathfinding, fasciculation, and blood vessel formation during peripheral nervous system (PNS) development. We confirmed a prior report (Exp. Neurol. 172 (2001) 398) that VEGF mRNA levels rise during Wallerian degeneration in the PNS and herein demonstrate that NP-1, NP-2, Sema3A, and Sema3F mRNA levels increase in peripheral nerves distal to a transection or crush injury. In a sciatic nerve crush model, in which axonal regeneration is robust, the highest levels of Sema3F mRNA below the injury site are in the epi- and perineurium. Our results suggest the possibility that the neuropilins and their semaphorin ligands serve to guide, rather than to impede, regenerating axons in the adult PNS.     

Evidence that Very Slow Wallerian Degeneration in C57BL/Ola Mice is an Intrinsic Property of the Peripheral Nerve

We have described a mutant mouse, C57BL/Ola, in which Wallerian degeneration following peripheral nerve transection is very slow. Our previous results suggested that recruited monocytes play a role in rapid Wallerian degeneration. The nature of the mutation in C57BL/Ola mice is not known and we have investigated whether the defect is intrinsic to the nerve or due to a defect in the circulating monocytes. We have made chimaeric mice in which bone marrow from histocompatible mice, with rapidly degenerating nerves and normal monocyte recruitment, was used to reconstitute irradiated C57BL/Ola mice and vice-versa. A substantial degree of donor repopulation of the hosts was confirmed by measures of the levels of glucose-phosphate isomerase alloenzymes in blood and tissue samples from the two different strains. The rate of degeneration of the transected sciatic nerve was found to be host-dependent, providing evidence that the mutation affects cell populations intrinsic to the nerve and not the circulating monocytes. We provide additional evidence that the peripheral nerves of C57BL/Ola mice are different from those of other mice as they degenerate at a slower rate in vitro.     

Very Slow Retrograde and Wallerian Degeneration in the CNS of C57BL/Ola Mice

Wallerian degeneration following peripheral nerve transection in C57BL/Ola mice is very slow in comparison to other strains of mice. We show that following optic nerve transection, the axons of retinal ganglion cells in C57BL/Ola mice undergo very slow Wallerian degeneration and that retrograde degeneration of the ganglion cell bodies is much slower than in other strains of mice. The results suggest that the gene product affecting Wallerian degeneration in the peripheral nervous system (PNS) also confers a greater resistance to degeneration on central nervous system (CNS) neurons.     

Expression changes and bioinformatic analysis of Wallerian degeneration after sciatic nerve injury in rat

Wallerian degeneration (WD) remains an important research topic. Many genes are differentially expressed during the process of WD, but the precise mechanisms responsible for these differentiations are not completely understood. In this study, we used microarrays to analyze the expression changes of the distal nerve stump at 0, 1, 4, 7, 14, 21 and 28 days after sciatic nerve injury in rats. The data revealed 6 076 differentially-expressed genes, with 23 types of expression, specifically enriched in genes associated with nerve development and axonogenesis, cytokine biosynthesis, cell differentiation, cytokine/chemokine production, neuron differentiation, cytokinesis, phosphorylation and axon regeneration. Kyoto Encyclopedia of Genes and Genomes pathway analysis gave findings related mainly to the MAPK signaling pathway, the Jak-STAT signaling pathway, the cell cycle, cytokine-cytokine receptor interaction, the p53 signaling pathway and the Wnt signaling pathway. Some key factors were NGF, MAG, CNTF, CTNNA2, p53, JAK2, PLCB1, STAT3, BDNF, PRKC, collagen II, FGF, THBS4, TNC and c-Src, which were further validated by real-time quantitative PCR, Western blot, and immunohistochemistry. Our findings contribute to a better understanding of the functional analysis of differentially-expressed genes in WD and may shed light on the molecular mechanisms of nerve degeneration and regeneration.     

cAMP cascade (PKA, Epac, adenylyl cyclase, Gi, and phosphodiesterases) regulates myelin phagocytosis mediated by complement receptor-3 and scavenger receptor-AI/II in microglia and macrophages

The removal by phagocytosis of degenerated myelin is central for repair in Wallerian degeneration that follows traumatic injury to axons and in autoimmune demyelinating diseases (e.g., multiple sclerosis). We tested for roles played by the cAMP cascade in the regulation of myelin phagocytosis mediated by complement receptor-3 (CR3/MAC-1) and scavenger receptor-AI/II (SRAI/II) separately and combined in mouse microglia and macrophages. Components of the cAMP cascade tested are cAMP, adenylyl cyclase (AC), Gi, protein kinase A (PKA), exchange protein directly activated by cAMP (Epac), and phosphodiesterases (PDE). PKA inhibitors H-89 and PKI(14-22) amide inhibited phagocytosis at normal operating cAMP levels (i.e., those occurring in the absence of reagents that alter cAMP levels), suggesting activation of phagocytosis through PKA at normal cAMP levels. Phagocytosis was inhibited by reagents that elevate endogenous cAMP levels to above normal: Gi-inhibitor Pertussis toxin (PTX), AC activator Forskolin, and PDE inhibitors IBMX and Rolipram. Phagocytosis was inhibited also by cAMP analogues whose addition mimics abnormal elevations in endogenous cAMP levels: nonselective 8-bromo-cAMP, PKA-specific 6-Benz-cAMP, and Epac-specific 8-CPT-2'-O-Me-cAMP, suggesting that abnormal high cAMP levels inhibit phagocytosis through PKA and Epac. Altogether, observations suggest a dual role for cAMP and PKA in phagocytosis: activation at normal cAMP levels and inhibition at higher. Furthermore, a balance between Gi-controlled cAMP production by AC and cAMP degradation by PDE maintains normal operating cAMP levels that enable efficient phagocytosis.