Axonal autophagy during regeneration of the rat sciatic nerve**★

BACKGROUND: The removal of degenerated axonal debris during Wallerian degeneration is very important for nerve regeneration. However, the mechanism by which debris is removed is not been completely understood. Considerable controversy remains as to the clearance pathway and cells that are involved. OBJECTIVE: To investigate axonal autophagy during removal of degenerated axonal debris by transecting the sciatic nerve in a rat Wallerian degeneration model. DESIGN, TIME AND SETTING: Experimental neuropathological analysis. The experiment was conducted at the Laboratory Animal Service Center of the Southern Medical University between January and June 2005. MATERIALS: Fifty-four adult, Wistar rats of either sex, weighing 180-250 g, were obtained from the Laboratory Animal Service Center of the Southern Medical University. Animals were randomly divided into nine groups of six rats. METHODS: Wallerian degeneration was induced by transecting the rat sciatic nerve, and tissue samples from the distal stump were obtained 0.2, 0.4, 1, 2, 3, 4, 7, 10, and 15 days post-transection. Ultrathin sections were prepared for electron microscopy to study ultrastructure and enzyme cytochemistry staining. MAIN OUTCOME MEASURES: Ultrastructure (axon body, autophagic body, and cystoskeleton) of axons and myelin sheaths observed with electron microscopy; acidic phosphatase activity detected by Gomori staining using electron microscopy. RESULTS: The major changes of degenerating axons after transection were axoplasm swelling and separation of axons from their myelin sheath between five hours and two days post-transection. At four days post-transection, the axoplasm condensed and axons were completely separated from the myelin sheath, forming dissociative axon bodies. Vacuoles of different sizes formed in axons during the early phase after lesion. Larger dissociative axon bodies were formed when the axons were completely separated from the myelin sheath during a late phase. The axolemma surrounding the axon body was derived from the neuronal cell membrane; the condensed axoplasm contained many autophagic vacuoles at all levels. A large number of neurofilaments, microtubules, and microfilaments were arranged in a criss-cross pattern. The autophagic vacuoles exhibited acidic phosphatase activity. Axonal bodies were absorbed after degradation from day 7 onwards, and macrophages were observed rarely in the formative cavity. CONCLUSION: The degenerating axons were cleared mainly by axonal autophagy and Schwann cell phagocytosis during regeneration of the rat sciatic nerve, and macrophages exhibited only an assisting function. Key Words: axon; autophagy; nerve regeneration     

Visualization of axonal degeneration after optic nerve lesion in rat by immunohistochemical labelling for myelin basic protein (MBP)

Immunohistochemical staining of myelin basic protein (MBP) was followed during axonal degeneration of rat retinal fibers within the first 3 weeks after injury. Wallerian degeneration of rat retinal fibers was elicited by unilateral transection or crush injury of optic nerve. MBP-labelled fibers in central retinal pathways and visual nuclei showed sequential changes of the myelin sheath, such as swelling at 1-2 days post lesion (dpi), granular staining at 4-8 dpi, and granular debris formation at 21 dpi. Consequently, immunostaining for MBP could be used to identify early stages of degenerating myelin and persisting myelin debris which is known to contain neurite growth inhibitors.     

Myelin sheath survival following axonal degeneration in doubly myelinated nerve fibers.

Axonal contact plays a critical role in initiating myelin formation by Schwann cells. However, recent studies of "double myelination" have indicated that myelin maintenance continues in Schwann cells completely displaced from physical contact with the axon. This raises the possibility either that diffusible trophic factors are produced by the axon, or that the axon is not required for myelin maintenance by these displaced Schwann cells. To test these hypotheses, the axons involved in double myelination in the mouse superior cervical ganglion (SCG) were transected surgically by a transganglionic lesion. The inferior pole of the SCG was resected to limit axonal regeneration. This method produced a typical Wallerian pattern of degeneration in the superior pole, without compromising the blood supply or introducing nonspecific trauma. EM analysis at 1 and 5 d postoperatively showed that initially the axon degenerated, followed by breakdown of the inner myelin sheath. In those configurations where the outer Schwann cell was only partly displaced from the axon, the outer myelin sheath degenerated simultaneously. However, in completely displaced internodes the outer sheath survived degeneration of the axon and inner sheath. Outer internodes remained intact for at least 5 weeks after transection (the longest time point in this study), at which time they enclosed reorganized processes of the inner Schwann cells, their basal lamina, and numerous collagen fibrils. Axonal regeneration within surviving outer internodes was rare and was characterized by the development of typical Remak ensheathment by the inner Schwann cells. We conclude that in the mouse SCG, myelin maintenance does not depend on the continued presence of the axon. These data suggest further that myelin breakdown in Wallerian degeneration may be initiated by mechanisms other than absence of a viable axon.     

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.     

Cell death of motoneurons in the chick embryo spinal cord. II. A quantitative and qualitative analysis of degeneration in the ventral root, including evidence for axon outgrowth and limb innervation prior to cell death.

Development of the ventral roots in the caudal half of the chick lumbar spinal cord (segments 26-29) was studied by electron microscopy. The ventral root fibers at 4, 5, 6, 7, 8, 9, and 13 days of incubation and at 1, 10 days and 5 weeks post-hatching were counted either from photomontages or directly in the electron microscope. In the chick, most, if not all, the fibers in the caudal ventral roots studied here probably arise from axons of motoneurons in the lateral and medial motor columns. At four days of incubation, there is an average of 800 axons per segment. The number increases very rapidly reaching a peak of 5,500 axons at day 5.5 In other words, by day 5.5 all the axons of motoneurons hav ealready reached the ventral root region. Between days 6 and 9, the number dreastically declines to 2,200 axons poer segment, a 57% reduction in ventral root fibers. After day 9, there is only aminor and rather slow additional loss of axons, reaching 1, 700 in the 13-day embryo and 1,500 in the 1-day post-hatching chick. In brief, during embryonic development about 71% of the axons are depleted in the ventral roots. Quantitatuve comparisons of Motoneurons in the lateral motor column (LMC) of segments 26-29 with the axon counts from the same segments have demonstrated: (a) that there is a massive natural cell loss in this region between days 5.5 and 9 amounting to 53%; (b) that axons are lost to the same extent as the motoneurons during this period, Resulting in a close to 1:1 relationship between the two by day 9. When horseradish peroxidase (HRP) was injected into the limb-buds of 5-day embryos, prior to the onset of massive cell death, virtually all motoneurons in the LMC were found to contain the HRP reaction product. Since approximately 50% of the cells present on day 5 typically degenerate by day 9, this finding, coupled with the observed close correspondence between axon and cell counts, strongly indicates that all motoneurons, even those destined to die, normally innervate the leg. Ultrastructural changes of motoneuron axons undergoing spontaneous degeneration in the ventral root were also described. The degenerating axons are found in the ventral root as early as the fourth day of incubation, although the number at this time is very low. More massive degeneration occurs between 5.5 and 9 days of incubation. The increrased number of degenerating axons in the ventral root during this period is in agreement with the increased number of degenerating cell bodies in the spinal lateral motor column at these same stages. Between day 4 and day 9, the degenerating axons are characterized by the presence of numerous vesiculated structures, membrane-bounded autophagic vacuoles, membranous lamellar figures and electron dense bodies in focal, swollen portions of the axon, as well as the disruption of the axolemma. The degeneration process seems to be due to progressive autolysis with the final axonal remnants being phagocytozed by the surrounding Schwann cells and some mononuclear leukocytes. Approximately 60% of the ventral root fibers completely disappear within three to four days, leaving very little evidence of axonal debris. We have found no differences in the details of axonal degeneration of ventral roots from limbbud removal embryos. The spontaneous degeneration of axons continues even after hatching but on a much reduced scale. The post-hatching degeneration is evidenced by the loosening of myelin sheath, shrinking of th axoplasm, an increase in both multivesicular bodies and lamellated dense bodies, and the disintergration of neurofilaments and neurotubules.     

Wallerian degeneration in the optic nerve of the wabbler-lethal (wl/wl) mouse.

Optic nerve pathology was studied in C57BL/6J wabbler-lethal (wl/wl) and control (+/+) mice at postnatal age of 4 weeks (P28). Qualitative light and ultrastructural pathology in wl/wl animals conformed to the criteria of primary axonal (Wallerian) degeneration. Most optic nerve axons in mutant animals appeared normal, as did oligodendroglia, the degree of myelination, the integrity and maturity of vascular elements, astroglia, and most myelin. Still, degenerating axons surrounded by somewhat normal myelin and axons with thickened myelin sheaths were prevalent in wl/wl mice. Dysmyelination or hypomyelination was not evident. At P28, pathology appeared more prominent in large diameter fibers. In the optic nerve of wl/wl mice, axonal degeneration preceded myelin disruption, adding this nerve to other previously reported systems undergoing Wallerian degeneration in this mutant.     

Relationship of Wallerian degeneration to regrowing axons.

Because Wallerian degeneration constitutes a highly significant and unavoidable position in the sequence of nervous system regeneration we have extended our observations regarding this process as it occurs in our cryogenic model of spinal cord injury in the rat. In previous studies we have observed essentially no growth into the Wallerian zone despite a significant amount of axonal regrowth through the injured region. In the present study we have examined by electron microscopy the Wallerian degeneration and its interface with the axons from the injured region. In the Wallerian region our observations have not only confirmed those of many previous investigators but have suggested an orderly sequence of changes during the first 60 days post-injury, beginning with relative tissue preservation followed by depletion of degenerating axoplasm, extracellular deposition of myelin and the development of a cellular reaction. Associated with the cellular reaction is the development of large astrocytic cells which can be found adjacent to unmyelinated and thinly myelinated axons. With this limited evidence of support for axonal growth in the Wallerian zone it is suggested that further expansion of this cellular matrix should provide significant support to regrowing axons.     

Delayed Macrophage Responses and Myelin Clearance during Wallerian Degeneration in the Central Nervous System: The Dorsal Radiculotomy Model

All aspects of Wallerian degeneration (WD)—axonal breakdown, glial and macrophage responses, and clearance of myelin debris—have generally been considered to occur more slowly in the central nervous system (CNS) than in the peripheral nervous system (PNS). We reevaluated this issue by comparing the temporal pattern of Wallerian degeneration in nerve fibers with segments extending through both the PNS and the CNS. The L₄, L₅, and L₆ dorsal roots in the rat were transected, and WD in the dorsal roots and the dorsal columns was compared at intervals up to 8 months, using electron microscopy and immunostaining to identify and characterize the different cell types. The initial breakdown of axoplasm was complete by 72 h both in the PNS and in the CNS portions of these axons. All other aspects of WD were strikingly delayed in the CNS when compared to those in the PNS. Macrophages (from the circulation) increased in number (Days 2-4 after axotomy) in the root. In contrast, although there was an early and transient period (peaking at Day 3) of microglial activation in the degenerating dorsal column, the appearance of round macrophages was delayed until Days 18-21. Both axonal debris and myelin debris were almost completely cleared by 30 days in the PNS, but remained over 90 days in the CNS. Axonal regeneration was vigorous in the dorsal root but these sprouts did not invade the dorsal columns. The dorsal root entry zone provided a sharp anatomic demarcation between the PNS and CNS patterns of Wallerian degeneration. These results suggest that circulating macrophages have ready access to degenerating peripheral nerves, but are largely or completely excluded from degenerating CNS tracts, so that the macrophages (that ultimately appear) originate primarily from the stellate microglia.     

PRIMARY DEMYELINATION IN CAT OPTIC NERVES ASSOCIATED WITH SURGICALLY INDUCED AXONAL DEGENERATION

Localized Wallerian degeneration was induced in cat optic nerves by the gentle scratching of the exposed retinas. At intervals ranging up to 103 days after operation, the cats were killed and microscopic examination of the optic nerves showed, in addition to axonal degeneration, the presence of both demyelinating and demyelinated normal axons. The tongues of oligodendroglial cytoplasm were still associated with these demyelinated axons. This phenomenon is considered to reflect a change in the homeostasis of the oligodendroglial cell imposed by degeneration of a few axons from a state of maintaining the myelin sheath to one of resorption from adjacent normal axons. No evidence for the involvement of microglia in this process was found. It is concluded also that oligodendrocytes alone can be responsible for the removal of myelin debris during Wallerian degeneration. This observation may be important to the understanding of certain demyelinating diseases of the central nervous system.     

Morphometric effects of vincristine on nerve regeneration in the rat

Administration of vincristine (200, 100 or 50 micrograms/kg/week) for 6 months during regeneration of the sciatic nerve after crush injury caused a dose-dependent reduction in nerve fibre size and failure of removal of myelin debris. Successfully regenerating neurites showed an unusual amount of shape distortion. The ratio of myelin sheath thickness to axon circumference was reduced, but the ratio of myelin sheath thickness to axon area was normal. Microtubule concentration was diminished in axons, but neurofilament density was unaffected. Unmyelinated axons were reduced in number but their axon diameter distribution was not affected. Fibres on the non-crushed side appeared normal. The toxicity of vincristine to regenerating nerves is probably related to increased blood-nerve permeability occurring both at the site of crush and along the degenerating nerve.