HISTOCHEMISTRY OF MYELIN–XV. CHANGES IN THE MYELIN PROTEINS OF THE PERIPHERAL NERVE UNDERGOING WALLERIAN DEGENERATION–ELECTROPHORETIC AND MICRODENSITOMETRIC OBSERVATIONS

The chronological order of changes in rat peripheral nerve proteins during Wallerian degeneration has been investigated by microdensitometric and electrophoretic techniques. Both methods revealed an early loss of myelin proteins. The histochemical microdensitometric study showed a very substantial early loss of stainable protein basic groups and a somewhat slower progressive loss of the major protein component of peripheral nerve myelin (the J band). The electrophoretic study showed an early loss of both the J band protein and the slower-moving basic protein band. The histochemical study also suggested that some cerebroside may be lost in the early stage of Wallerian degeneration. It is concluded that degradation of myelin proteins is an initial event in the process of myelin breakdown.     

Biochemical studies of myelin in Wallerian degeneration of rat optic nerve

Abstract— Wallerian degeneration of the optic nerves of the rat was induced by removal of the eyes. After 54, 66, 76 or 90 days of degeneration a myelin fraction of the nerves was obtained by the procedure of Laatsch et al. (1962). The yield of myelin from the degenerated nerves was decreased, but the isolated myelin appeared to be morphologically normal. The proportion of cholesterol in the myelin lipids was slightly increased, whereas that of the ethanolamineglycerophosphatides was decreased and galactolipids were normal. After one‘cycle’of myelin purification, the high-molecular-weight fraction formed a much greater percentage of the total protein in myelin isolated from degenerated optic nerves. After 2–3‘cycles’of purification, the distribution of protein in myelin isolated from degenerated and normal optic nerves was similar, an observation suggesting that the high-molecular-weight fraction in‘1-cycle myelin’from degenerated optic nerves may have been partly attributable to contamination. With the possible exception of ethanolamineglycerophosphatides, our data suggest that there was no preferential breakdown of myelin lipid constituents nor of protein constituents during Wallerian degeneration of rat optic nerve. As assessed by SDS-gel electrophoresis of the water-insoluble particulate fraction, the percentage of myelin protein was markedly decreased after 76 days of degeneration. However, the major myelin protein constituents in this fraction (the two basic proteins and proteolipid protein) appeared to decrease in the same relative proportions.     

Myelin Proteins, Glycoproteins, and Myelin-Related Enzymes in Experimental Demyelination of the Rabbit Optic Nerve: Sequence of Events

Wallerian degeneration of the rabbit optic nerve was investigated by the technique of retinal ablation which precludes edema, hemorrhage, or macrophage infiltration. After 8 days of degeneration, marked degradation of axons and some myelin abnormalities appeared in the optic nerve, optic chiasma, and optic tract. Myelin lesions were maximal 32 days after retinal destruction. The amount of material stained with a myelin dye decreased drastically between 32 and 90 days after the operation. Biochemical parameters gave the following sequence of events. The concentration of the major periodic acid--Schiff staining glycoproteins was decreased after 2 days, and 6 days later the presence of cholesterol esters was detected in the optic tissue. After 16 days of Wallerian degeneration, the specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase not associated with myelin decreased, indicating a possible de-differentiation of oligodendrocytes. Degradation of myelin basic protein became significant at 32 days and the amount of myelin isolated decreased later. The loss of myelin basic protein coincided with a reduction of myelin periodicity as measured in purified fractions by electron microscopy. These results show that secondary myelin destruction in the absence of edema, hemorrhage, or macrophages is a very slow process, and in this situation myelin undergoes a selective and sequential loss of its constituents.     

FINE STRUCTURAL LOCALIZATION OF CHOLESTEROL-1,2-3H IN DEGENERATING AND REGENERATING MOUSE SCIATIC NERVE

The localization of ³H-labeled cholesterol in nerves undergoing degeneration and regeneration was studied by radioautography at the electron microscope level. Two types of experiments were carried out: (a) Cholesterol-1,2-³H was injected intraperitoneally into suckling mice. 5 wk later, Wallerian degeneration was induced in the middle branch of the sciatic nerve, carefully preserving the collateral branches. The animals were then sacrificed at various times after the operation. During degeneration, radioactivity was found over myelin debris and fat droplets. In early stages of regeneration, radioactivity was found in myelin debris and regenerating myelin sheaths. Afterwards, radioactivity was found predominantly over the regenerated myelin sheaths. Radioactivity was also associated with the myelin sheaths of the unaltered fibers, (b) Wallerian degeneration was induced in the middle branch of the sciatic nerves of an adult mouse, preserving the collateral branches. Cholesterol-1,2-³H was injected 24 and 48 hr after the operation and the animal was sacrificed 6 wk later. Radioactivity was found in the myelin sheaths of the regenerated and unaltered fibers. The results from these experiments indicate that: (a) exogenous cholesterol incorporated into peripheral nerve during myelination remains within the nerve when it undergoes degeneration. Such cholesterol is kept in the myelin debris as an exchangeable pool from which it is reutilized for the formation of the newly regenerating fibers, especially myelin. (b) exogenous cholesterol incorporated into the nerves at the time that degeneration is beginning is also used in the formation of new myelin sheaths during regeneration, (c) mature myelin maintains its ability to incorporate cholesterol.     

Myelin sheath survival after guanethidine-induced axonal degeneration

Membrane-membrane interactions between axons and Schwann cells are required for initial myelin formation in the peripheral nervous system. However, recent studies of double myelination in sympathetic nerve have indicated that myelin sheaths continue to exist after complete loss of axonal contact (Kidd, G. J., and J. W. Heath. 1988. J. Neurocytol. 17:245-261). This suggests that myelin maintenance may be regulated either by diffusible axonal factors or by nonaxonal mechanisms. To test these hypotheses, axons involved in double myelination in the rat superior cervical ganglion were destroyed by chronic guanethidine treatment. Guanethidine-induced sympathectomy resulted in a Wallerian-like pattern of myelin degeneration within 10 d. In doubly myelinated configurations the axon, inner myelin sheath (which lies in contact with the axon), and approximately 75% of outer myelin sheaths broke down by this time. Degenerating outer sheaths were not found at later periods. It is probably that outer sheaths that degenerated were only partially displaced from the axon at the commencement of guanethidine treatment. In contrast, analysis of serial sections showed that completely displaced outer internodes remained ultrastructurally intact. These internodes survived degeneration of the axon and inner sheath, and during the later time points (2-6 wk) they enclosed only connective tissue elements and reorganized Schwann cells/processes. Axonal regeneration was not observed within surviving outer internodes. We therefore conclude that myelin maintenance in the superior cervical ganglion is not dependent on direct axonal contact or diffusible axonal factors. In addition, physical association of Schwann cells with the degenerating axon may be an important factor in precipitating myelin breakdown during Wallerian degeneration.     

Zur Mechanik der Markballenbildung bei der Wallerschen Degeneration

Summary Wallerian degeneration was studied in frog single fibers. A new preparation for microscopic observation of peripheral motor fibers in living Rana esculenta was used. The authors studied proximally transected groups of fibers using the polarization technique.The results confirmed that the axon destruction preceds the breakdown of the myelin sheath, and the Schmidt-Lanterman incisures play an important role in the formation of ovoids. Details of the ovoid formation are demonstrated in serial photographs. It is suggested that the incisures of Schmidt-Lanterman are pathways for electrolyte and water transport in Wallerian degeneration.     

Phagocytosis of degenerating myelin in transected feline optic nerve: an immunohistochemical study

The phagocytic activity of neuroglial cells in adult feline degenerating optic nerve was investigated by immunocytochemistry at both light and electron microscopy levels. Degeneration was initiated by unilateral eye enucleation and the segment distal to the transection showing true Wallerian degeneration was examined. Following enucleation, twelve adult domestic cats were examined over a period of seven to 215 days. All cases showed slow clearance of myelin debris and absence of proliferating monocytes throughout the post-enucleation period. All phagocytic cells present were neuroglial cells, and many of these cells expressed oligodendroglial antigens. These findings demonstrate the persistence of an active population of oligodendrocytes that might play an additional functional role during Wallerian degeneration of feline optic nerve.     

Phagocytosis of degenerating myelin in transected feline optic nerve: an immunohistochemical study

The phagocytic activity of neuroglial cells in adult feline degenerating optic nerve was investigated by immunocytochemistry at both light and electron microscopy levels. Degeneration was initiated by unilateral eye enucleation and the segment distal to the transection showing true Wallerian degeneration was examined. Following enucleation, twelve adult domestic cats were examined over a period of seven to 215 days. All cases showed slow clearance of myelin debris and absence of proliferating monocytes throughout the post-enucleation period. All phagocytic cells present were neuroglial cells, and many of these cells expressed oligodendroglial antigens. These findings demonstrate the persistence of an active population of oligodendrocytes that might play an additional functional role during Wallerian degeneration of feline optic nerve.     

Phagocytosis of degenerating myelin in transected feline optic nerve: an immunohistochemical study.

The phagocytic activity of neuroglial cells in adult feline degenerating optic nerve was investigated by immunocytochemistry at both light and electron microscopy levels. Degeneration was initiated by unilateral eye enucleation and the segment distal to the transection showing true Wallerian degeneration was examined. Following enucleation, twelve adult domestic cats were examined over a period of seven to 215 days. All cases showed slow clearance of myelin debris and absence of proliferating monocytes throughout the post-enucleation period. All phagocytic cells present were neuroglial cells, and many of these cells expressed oligodendroglial antigens. These findings demonstrate the persistence of an active population of oligodendrocytes that might play an additional functional role during Wallerian degeneration of feline optic nerve.     

Degenerating Myelin: Comparative Histochemical Studies Using Classical Myelin Stains and an Improved Marchi Technique Minimizing Artifacts

In this study on degenerating myelin, comparisons were made between different stains including Marchi, regular lipid, and Weil's stain for myelin sheaths. An improved technique was devised which minimized artifacts regularly seen in the classical stains for myelin and for degenerating myelin. An artifact referred to as “pseudo-Marchi dust” is eliminated by the method of handling and fixing brain and spinal cord tissue. Some discussion is included on the solubilities of various tissue constituents which may be affected by prior freezing of unreacted tissue. In view of this, the varying sequence of events in degenerative processes of the central nervous system (CNS) brought about by different etiologic factors, such as Wallerian degeneration, phalaris staggers and enzootic ataxia, might be re-evaluated by the use of several interrelated staining methods. The CNS diseases studied here using a number of staining techniques are considered to be the result of local toxic conditions rather than due to tract interruption. The present study suggests evaluations of the stained sections in terms of the histochemical reactions involved. The degenerative sequence in phalaris staggers appears to be different from that seen in enzootic ataxia in that tissues from sheep with phalaris staggers showing early and advanced neurological changes have differing patterns of lipid stainability and the combined staining procedures applied to CNS tissues from sheep with enzootic ataxia show characteristics more like the CNS tissues from sheep with advanced phalaris staggers than from early phalaris staggers. Characteristics of both conditions differ from the histological pattern reported for classical Wallerian degeneration.     

Schwann cell autophagy,myelinophagy, initiates myelin clearance from injured nerves

Although Schwann cell myelin breakdown is the universal outcome of a remarkably wide range of conditions that cause disease or injury to peripheral nerves, the cellular and molecular mechanisms that make Schwann cell–mediated myelin digestion possible have not been established. We report that Schwann cells degrade myelin after injury by a novel form of selective autophagy, myelinophagy. Autophagy was up-regulated by myelinating Schwann cells after nerve injury, myelin debris was present in autophagosomes, and pharmacological and genetic inhibition of autophagy impaired myelin clearance. Myelinophagy was positively regulated by the Schwann cell JNK/c-Jun pathway, a central regulator of the Schwann cell reprogramming induced by nerve injury. We also present evidence that myelinophagy is defective in the injured central nervous system. These results reveal an important role for inductive autophagy during Wallerian degeneration, and point to potential mechanistic targets for accelerating myelin clearance and improving demyelinating disease.     

Wallerian degeneration in central nervous system: dynamic associations between diffusion indices and their underlying pathology

Background

Although diffusion tensor imaging has been used to monitor Wallerian degeneration, the exact relationship between the evolution of diffusion indices and its underlying pathology, especially in central nervous system, remains largely unknown. Here we aimed to address this question using a cat Wallerian degeneration model of corticospinal tract.

Methodology/Principal Findings

Twenty-five domestic mature Felis catus were included in the present study. The evolution of diffusion indices, including mean diffusivity (MD), fractional anisotropy (FA), primary (λ1) and transverse eigenvalues (λ23) of the degenerated corticospinal tract, were observed at baseline (before modeling) and at 2, 4, 6, 8, 10, 15, 20, 25, 30, 45 and 60 days after modeling in 4 cats. Pathological examinations were performed at eight time points mentioned above. Wallerian degeneration can be detected as early as the 2nd day after modeling by both diffusion tensor imaging and pathology. According to the evolution of diffusion indices, Wallerian degeneration can be classified into 2 stages. During the early stage (within 8 days after modeling), progressive disintegration of axons and myelin sheaths underlies the decreases in FA and λ1 and the increase in λ23. However, during the late stage (after 8 days), the gradual increases in FA, MD and λ1 and the unchanged λ23 seem to be a comprehensive reflection of the pathological processes including microglia activation, myelin clearance, and astrocytosis.

Conclusions/Significance

Our findings help the understanding of the altered diffusion indices in the context of pathology and suggest that diffusion tensor imaging has the potential to monitor the processes of Wallerian degeneration in the central nervous system in vivo after acute damage.     

Histochemistry of myelin. XI. Loss of basic protein in early myelin breakdown and multiple sclerosis plaques

Synopsis the early stages of Wallerian degeneration in peripheral nerves are accompanied by loss of a trypanophilic, trypsin-digestible basic protein from myelin. This loss of basic protein is ascribed to the activity of proteolytic enzymes. The reduced trypanophilia in degenerating nerves could not be attributed to loss of lipid. Likewise, the tryptophan-rich trypsin-resistant neurokeratin component of peripheral nerve myelin showed no change in the first week of degeneration. Loss of basic protein has been observed in and surrounding plaques of multiple sclerosis. We infer that digestion of basic protein would lead to the release of the encephalitogenic antigen contained therein.Research Associate supported by the British Multiple Sclerosis Society.     

Incorporation of fucose and leucine into PNS myelin proteins in nerves undergoing early Wallerian degeneration

The simultaneous incorporation of [³H]fucose and [1-¹⁴C]leucine into normal rat sciatic nerve was examined using an in vitro incubation model. A linear rate of protein precursor uptake was found in purified myelin protein over 1/2–6 hr of incubation utilizing a supplemented medium containing amino acids. This model was then used to examine myelin protein synthesis in nerves undergoing degeneration at 1–4 days following a crush injury. Data showed a statistically significant decrease in the ratio of fucose to leucine at 2, 3, and 4 days of degeneration, which was the consequence of a significant increase in leucine uptake. These results, plus substantial protein recovery in axotomized nerves, are indicative of active synthesis of proteins that purify with myelin during early Wallerian degeneration.     

Adenosine 5′-Triphosphate (ATP) Inhibits Schwann Cell Demyelination During Wallerian Degeneration

Adenosine 5′-triphosphate (ATP) is implicated in intercellular communication as a neurotransmitter in the peripheral nervous system. In addition, ATP is known as lysosomal exocytosis activator. In this study, we investigated the role of extracellular ATP on demyelination during Wallerian degeneration (WD) using ex vivo and in vivo nerve degeneration models. We found that extracellular ATP inhibited myelin fragmentation and axonal degradation during WD. Furthermore, metformin and chlorpromazine, lysosomal exocytosis antagonists blocked the effect of ATP on the inhibition of demyelination. Thus, these findings indicate that ATP-induced-lysosomal exocytosis may be involved in demyelination during WD.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

Wallerian degeneration in rabbit tibial nerve: changes in amounts of the S-100 protein

Abstract— —A soluble protein (S-100) which is unique to the nervous system was measured in rabbit tibial nerve at 0, 3, 7, 14, 21, and 28 days of degeneration. Amounts of S-100 in the degenerated peripheral segment of the transected nerve fell progressively during degeneration to 2 per cent of that measured in the corresponding portion of nerve taken from control rabbits 28 days postoperatively. Total soluble proteins increased 42 per cent during this time. Levels of S-100 and total soluble proteins remained unchanged in non-degenerated nerve segments from experimental and control rabbits. Correlations of amounts of S-100 measured in the study reported here with cellular changes demonstrated by other investigators to characterize Wallerian degeneration in peripheral nerve suggest that the S-100 protein is localized primarily in axons rather than in Schwann cells or myelin.     

Non-glial phagocytes within the degenerating optic nerve of the newt (Triturus viridescens).

Two non-glial phagocytes were found to participate along with ependymoglial cells in Wallerian degeneration of the severed optic nerve of the newt (Triturus viridescens). The first type of non-glial cell (polymorphonuclear phagocyte) was positively identified as a neutrophil and participates in the early stages of degeneration. Cells of this type make a brief appearance, reaching a peak by the second postoperative day (2 p.o.d.), and quickly diminish until few can be found by 4 p.o.d. Neutrophils invade the degenerating optic nerve from surrounding connective tissue spaces, most likely, through channels which penetrate the nerve parenchyma. The second type of non-glial cell is an invading mononuclear phagocyte which exhibits characteristics of microglial cells reported in other vertebrate species. Such cells appear in the nerve much later than the neutrophils and towards the end of Wallerian degeneration (6-10 p.o.d.). Their mode of entry and exit appears to be the same as that reported for neutrophils. The neutrophils and microglial-like, mononuclear phagocytes may serve to supplement the histolytic action of the ependymoglial cells, picking up scattered fragments of degenerating myelin and axons.     

Incorporation of plasma radiophosphate into the inorganic, organic acid-soluble and phospholipid phosphate fractions of the normal rabbit sciatic nerve and during Wallerian degeneration]

Inorganic phosphate exchanges between plasma and sciatic nerve have been measured in the rabbit using a 32PO4 tracer technique. Inorganic phosphate is taken up at the rate of 0.13 microng per hour and per 100 mg fresh weight. Incorporation of plasma radiophosphate is markedly increased into the inorganic and organic acid soluble phosphate fractions of the distal part of the sectioned sciatic nerve. This increase is already signficant within one hour after surgical division, spreading at least 3 cm distally within 6 hours. This high level of incorporation persists until the 29th day of degeneration. These results favour the hypothesis that the axonal continuity maintains the metabolic activity of the Schwann cells at an inframaximal level. We confirm the rapid decrease in total phospholipid concentration in the nerve undergoing Wallerian degeneration as well as the marked increase in their specific activity. We show however that this increase in specific activity is due partly to the increased specific activities of the precursors (organic acid soluble phosphates), partly to the disappearance of a metabolically insert pool (myelin phospholipids). The Schwann cells of the nerve undergoing Wallerian degeneration do not have a more active phospholipid metabolism than their normal counterparts.