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.     

The role of Schmidt-Lanterman incisures in Wallerian degeneration. I. A quantitative teased fibre study

It is conventionally accepted that during the early stages of Wallerian degeneration of myelinated peripheral nerve fibres Schmidt-Lanterman incisures represent the sites at which the myelin sheath, together with enclosed axoplasm, is segmented into myelin ovoids. This mechanism is considered by some authors to be facilitated by the progressive intercalation of additional incisures in order to allow the later division of primary ovoids. We have demonstrated that this reported increase in the number of incisures is a misinterpretation of the changes occurring. By 36 h after crush of the rat sural nerve most myelinated fibres showed segmentation at incisures to form myelin ovoids. At 12 h and 24 h after crush, however, no ovoids were apparent and the number of incisures present was determined from teased fibres by light microscopy using oil immersion. There was no increase in the number of incisures either internodally or paranodally at 12 h and 24 h compared with a normal control population of fibres. However at 12 h, and to a greater extent at 24 h, incisures were more readily apparent than in normal fibres. It is likely, therefore, that previous reports have confused an increase in the number of incisures with an increase in their perceptibility resulting from their progressive dilatation.     

Wallerian degeneration in human nerves: serial electrophysiological studies.

After nerve transection, the distal stump undergoes Wallerian degeneration (WD). Little information is available concerning sequential changes in nerve conduction measurements during WD in humans. Five patients with nerve injuries were studied temporally. Motor-evoked amplitudes were reduced by 50% at 3 to 5 days after injury; the response was absent by day 9. Sensory-evoked amplitudes were reduced by 50% at 7 days after injury; the response was absent by day 11. Sensory and motor nerves with shorter distal stumps showed earlier loss of amplitude than did those with longer distal stumps. Denervation potentials were seen 10 to 14 days after injury. Our results suggest that WD occurs earlier if the distal stump is shorter, and that motor-evoked responses are affected earlier than sensory-evoked responses. The time-lag between the loss of the motor-evoked response and the appearance of denervation potentials, the latter coinciding with reduction of sensory evoked responses, suggests that failure of neuromuscular transmission precedes axonal loss during WD.     

Oligodendrocyte survival in Wallerian degeneration

Summary The long-term survival of oligodendrocytes in the absence of axons in adult animals was studied following Wallerian degeneration of the optic nerves of adult rats for periods up to 22 months. In contrast to the findings in development and in young animals, large numbers of oligodendrocytes survived during this time period even when deprived of axonal stimuli. The morphological phenotype of many of these cells differed from those of normal oligodendrocytes, and their oligodendrocytic nature was confirmed at a light microscopic level using antibodies to carbonic anhydrase, and at an electron microscopic level by using antibodies to myelin oligodendrocyte glycoprotein. Although there did appear to be some loss of oligodendrocytes over time, a large proportion appeared to remain intact. Many of the remaining cells and processes appeared to resemble resting or quiescent cells. The survival of these cells demonstrates the differential susceptibility of mature and developing oligodendrocytes to loss of axonal stimulus and also indicates a possible reserve capacity for repair following central nervous system injury.Supported by a grant MA 5818 from the Medical Research Council of Canada     

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.     

The blood-nerve barrier in Wallerian degeneration: a sequential long-term study

The blood-nerve barrier (BNB) for serum proteins was studied after a crush lesion of the murine sciatic nerve or after transsection with persistent Wallerian degeneration. Using single intraperitoneal injections of biotinylated human albumin, transferrin, IgG, and complement components as tracers, the integrity of the BNB during degeneration and regeneration was determined over time. In Wallerian degeneration induced by crush the BNB became increasingly leaky, with a maximum in the distal stump 8 days after crush (i.e., during early regeneration). When regeneration potentials could first be elicited from the small foot muscles and when thinly myelinated nerve fibers were present, the BNB gradually regained its barrier function and was nearly intact on day 30 after crush. After transsection breakdown of the BNB persisted beyond 30 days. The BNB leakage may foster repair by allowing exchange of trophic factors of large molecular size during nerve regeneration.     

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     

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.     

The spatio-temporal pattern of Wallerian degeneration in the Rhesus monkey optic nerve

Summary Three patterns of degeneration were distinguished in the Rhesus monkey optic nerve following eye enucleation. (1) A traumatic zone, extending for 1 to 2 mm from the site of transection and characterized by a rapid infiltration of haematogenous macrophages, the rapid degeneration and removal of all neural elements and the formation of astrocytic scar tissue within 35 days. (2) A conical zone, closely associated with the central retinal blood vessels, in which the pattern of degeneration was similar to the traumatic zone except that the onset was delayed and the removal of debris was slower. There was extensive vesiculation of myelin sheaths in these two zones which was indicative of haematogenous cell infiltration. (3) The remainder of the nerve underwent classical Wallerian degeneration in which endogenous cells slowly phagocytosed the degenerating nerve fibres. These observations are considered relevant to the controversy concerning the identification of the macrophage involved in Wallerian degeneration; many studies have described the macrophage within the area of trauma rather than the macrophage involved at some distance from the site of trauma.Supported by Grants 692-B-2 from the National Multiple Sclerosis Society; NS 02255 and NS 08180 from the National Institutes of Health and a grant from the Alfred P. Sloan Foundation     

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.     

Electrophysiologic changes accompanying Wallerian degeneration in frog sciatic nerve

The time course of the physiological changes accompanying Wallerian degeneration in the frog is markedly prolonged in comparison with that in mammals. Following transection of frog sciatic nerve, stimulation of the distal segment results in muscle contraction of normal amplitude through day 4, after which tension and EMG signals decline rapidly to levels that are undetectable by day 7. The compound action potential in the severed nerve continues for a much longer period of time, however, persisting as long as approximately 6 weeks. The amplitude remains at normal levels for nearly 3 weeks, after which it declines progressively, approaching zero at approximately 6 weeks. Conduction velocity remains normal in both alpha- and beta-fibers for approximately 3 weeks; thereafter, velocity in the alpha-fibers declines to approximately 71% of normal, and beta-fiber conduction is no longer detectable. Contraction could be elicited by direct stimulation of the muscle during the entire 6-week period of the study. The failure of nerve-mediated muscle activity in the face of persistent nerve conduction could be the result of either a defect in the most distal portions of the nerve fibers or a defect in neuromuscular transmission. These results suggest that EMG evaluation may be more sensitive than nerve conduction studies for clinical assessment of axonopathy at early stages.     

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)     

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.     

Wallerian degeneration of peripheral nerve

Summary The age-dependent loss of the major peripheral nerve lipids (cholesterol, phospholipid, and total galactolipid) was quantitated over a period of 9 weeks of Wallerian degeneration induced by surgical transection of rabbit sciatic nerves in animals of several ages. Proportionate losses of these lipids were determined by calculating the content of each lipid on a per nerve and on a per gram fresh weight basis remaining after a given period of Wallerian degeneration as a percent of original normal values at several times following surgery. The proportionate loss of each lipid from the distal stump was the most prompt and the most complete in nerves transected at 2 weeks of age, and the least in nerves transected at 20 weeks of age. The prompter clearance of these lipids from younger than older degenerating nerve gives convincing evidence that the suggestion from light-microscopic studies of faster clearance of neural debris in younger than in older animals is correct. A possible relationship between these biochemical findings and the phenomenon of greater functional recovery from peripheral nerve injury in younger than in older subjects is discussed.Supported by an N.I.H. grant (NS-10165)     

Phagocytosis in the rat optic nerve following Wallerian degeneration

Summary Unilateral enucleation of the eye in adult male rats was performed in an attempt to resolve the long-standing controversy as to the nature of the phagocytic cells during Wallerian degeneration in the central nervous system. Previously both resident microglia and circulating monocytes, as well as oligodendrocytes, have all been considered to be the phagocytic cells. In these present experiments macrophages and microglia were studied using lectin histochemistry for Griffonia simplicifolia agglutinin and the monoclonal antibody ED1 at light microscopic level. Oligodendrocytes were demonstrated ultrastructurally using immunohistochemistry with monoclonal antibodies against myelin oligodendrocyte glycoprotein (MOG). Ultrastructural examination of the degeneration optic nerves confirmed longstanding reports of the slow nature of breakdown in the adult central nervous system. During the early periods of breakdown, starting at 1 week and continuing to 1 or 2 months, it was difficult to type, on ultrastructural examination alone, the nature of all the cells undergoing phagocytosis, but many of them resembled microglia/macrophages. Myelin debris cleared very slowly and could still be recognised prominently in the nerve up to 22 months post-enucleation. Lectin and immunochemical examination showed that the early major phagocytic component of phagocytosis was carried out by macrophages, probably both circulating and resident. In addition, however, myelin and axonal debris was taken up or retracted into oligodendrocyte processes, which were stained with antibodies to MOG. This oligodendrocyte component appeared to be small in relationship to the overall degree of debris. Phagocytosis in the optic nerve, therefore, is in many respects similar to that in the peripheral nervous system, which has been shown to be carried out mainly by circulating macrophages, with only a minor role played by the Schwann cell.Supported by a grant MA 5818 from the Medical Research Council of Canada     

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.     

Adrenergic innervation of blood vessels in rat tibial nerve during Wallerian degeneration

Summary Adrenergic innervation of blood vessels in rat tibial nerve during Wallerian degeneration was examined, using the formaldehyde-induced histo-fluorescence method. The left sciatic nerve was transected at the level of the sciatic notch, whereas the right sciatic nerve was left intact and used as control. At 1, 3, 7, 14, 42, 56 or 84 days after transection, the tibial nerves of the transected and contralateral sides were exposed. Pieces of each nerve were used for light microscopy or for examination of adrenergic innervation with the fluorescence microscope. One day after transection, no adrenergic nerve fiber was observed in the endoneurium of the transected nerve. After 3 days, adrenergic innervation of small-and medium-sized arterioles in the epi-perineurium was absent, and after 7 days no fibers were visible around large arterioles. Fluorescent fibers were not detected even at 84 days post-surgery. It is concluded that adrenergic innervation of blood vessels in the rat tibial nerve is irreversibly lost after permanent axotomy, and that adrenergic regulation of nerve blood flow may also be lost.