Expression and regulation of neurotrophins in the nondegenerate and degenerate human intervertebral disc

Introduction

The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been identified in the human intervertebral disc (IVD) and have been implicated in the mechanisms associated with nerve ingrowth and nociception in degeneration of the IVD. The aim of the current study was to investigate an association between neurotrophin expression in the IVD and the severity of disc degeneration, including the effect of disc-related proinflammatory cytokines on neurotrophin and neuropeptide expression in cells derived from the human IVD.

Methods

Immunohistochemical analysis was performed to examine the expression of NGF, BDNF and their high-affinity receptors Trk-A and Trk-B in human IVD samples, divided into three categories: non-degenerate, moderate degeneration and severe degeneration. In order to study the effect of disc-related cytokines on neurotrophin/neuropeptide gene expression, nucleus pulposus cells derived from non-degenerate and degenerate IVD samples were seeded in alginate and were stimulated with either IL-1β or TNFα for 48 hours. RNA was extracted, cDNA was synthesised and quantitative real-time PCR was performed to examine the expression of NGF, BDNF and substance P.

Results

Immunohistochemistry showed expression of NGF and BDNF in the native chondrocyte-like cells in all regions of the IVD and in all grades of degeneration. Interestingly only BDNF significantly increased with the severity of degeneration (P < 0.05). Similar expression was observed for Trk-A and Trk-B, although no association with disease severity was demonstrated. In cultured human nucleus pulposus cells, stimulation with IL-1β led to significant increases in NGF and BDNF gene expression (P < 0.05). Treatment with TNFα was associated with an upregulation of substance P expression only.

Conclusion

Our findings show that both the annulus fibrosus and nucleus pulposus cells of the IVD express the neurotrophins NGF and BDNF, factors that may influence and enhance innervation and pain in the degenerate IVD. Expression of Trk-A and Trk-B by cells of the nondegenerate and degenerate IVD suggests an autocrine role for neurotrophins in regulation of disc cell biology. Furthermore, modulation of neurotrophin expression by IL-1β and modulation of substance P expression by TNFα, coupled with their increased expression in the degenerate IVD, highlights novel roles for these cytokines in regulating nerve ingrowth in the degenerate IVD and associated back pain.     

Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-β

Background

Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-β could target to nerve cells and improve nerve regeneration.

Methods

Laminin-binding assay and sustained release assay of NGF-β fused with NtA (LBD-NGF) from laminin in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested.

Findings

LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages.

Conclusion

Fused with NtA, NGF-β could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries.     

Addition of Exogenous NAD+ Prevents Mefloquine-Induced Neuroaxonal and Hair Cell Degeneration through Reduction of Caspase-3-Mediated Apoptosis in Cochlear Organotypic Cultures

Background

Mefloquine is widely used for the treatment of malaria. However, this drug is known to induce neurological side effects including depression, anxiety, balance disorder, and sensorineural hearing loss. Yet, there is currently no treatment for these side effects.

Principal Findings

In this study, we show that the coenzyme NAD⁺, known to play a critical role in maintaining the appropriate cellular redox environment, protects cochlear axons and sensory hair cells from mefloquine-induced degeneration in cultured rat cochleae. Mefloquine alone destroyed hair cells and nerve fiber axons in rat cochlear organotypics cultures in a dose-dependent manner, while treatment with NAD⁺ protected axons and hair cells from mefloquine-induced degeneration. Furthermore, cochlear organs treated with mefloquine showed increased oxidative stress marker levels, including superoxide and protein carbonyl, and increased apoptosis marker levels, including TUNEL-positive nuclei and caspases-3. Treatment with NAD⁺ reduced the levels of these oxidative stress and apoptosis markers.

Conclusions/Significance

Taken together, our findings suggest that that mefloquine disrupts the cellular redox environment and induces oxidative stress in cochlear hair cells and nerve fibers leading to caspases-3-mediated apoptosis of these structures. Exogenous NAD⁺ suppresses mefloquine-induced oxidative stress and prevents the degeneration of cochlear axons and sensory hair cells caused by mefloquine treatment.     

Metamorphosis of the marine gastropod Phestilla Sibogae Bergh (nudibranchia: aeolidacea). I. Light and electron microscopic analysis of larval and metamorphic stages

The structure and fate of transitory larval organs (velum, shell, operculum, retractor muscles, part of the epidermis) of Phestilla sibogae Bergh were studied before, during, and after metamorphosis with both light and electron microscopy to elucidate the morphology of these organs and the mechanisms by which they are lost.Loss of the velar lobes is the first morphological sign of metamorphosis, and involves selective dissociation and subsequent ingestion of the ciliated velar cells; the remaining aggregate of supportive cells is apparently incorporated into cephalic epidermis. Attachment of the larval body to shell and operculum is primarily at sites of retractor muscle insertions; once the velum is gone, the attachment between shell and larval body is lost and the shell is cast off as the visceral organs exit through the shell aperture. Merger of visceral and cephalopedal elements results in flattening of the postlarval body and reorientation of internal organs. Simultaneously, a rapid spreading of epipodial epidermis over the lateral, dorsal, and posterior sides of the body produces the definitive integument. The squamous cells which comprise the larval perivisceral epidermis are pushed ahead of the definitive epidermis and are seen shortly after the shell is cast as a constricted aggregate of cells on the posterior end of the body. Autolysis of the left and right retractor muscles begins during metamorphosis and no trace of them is left after 24 to 48 h. The metapodial mucous glands which hypertrophy before metamorphosis are also lost within 48 h following exit of the post larva from the shell. Metamorphosis produces a detorsion caused in part by muscular action and in part by continuing growth and development.     

Expression and regulation of neurotrophic and angiogenic factors during human intervertebral disc degeneration

Introduction

The degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells.

Methods

Quantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1β) stimulated NP cells.

Results

Initial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1β induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1β. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1β and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1β. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines.

Conclusions

The release of cytokines, in particular IL-1β during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1β is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.     

Scanning laser optical tomography resolves structural plasticity during regeneration in an insect brain

Background

Optical Projection Tomography (OPT) is a microscopic technique that generates three dimensional images from whole mount samples the size of which exceeds the maximum focal depth of confocal laser scanning microscopes. As an advancement of conventional emission-OPT, Scanning Laser Optical Tomography (SLOTy) allows simultaneous detection of fluorescence and absorbance with high sensitivity. In the present study, we employ SLOTy in a paradigm of brain plasticity in an insect model system.

Methodology

We visualize and quantify volumetric changes in sensory information procession centers in the adult locust, Locusta migratoria. Olfactory receptor neurons, which project from the antenna into the brain, are axotomized by crushing the antennal nerve or ablating the entire antenna. We follow the resulting degeneration and regeneration in the olfactory centers (antennal lobes and mushroom bodies) by measuring their size in reconstructed SLOTy images with respect to the untreated control side. Within three weeks post treatment antennal lobes with ablated antennae lose as much as 60% of their initial volume. In contrast, antennal lobes with crushed antennal nerves initially shrink as well, but regain size back to normal within three weeks. The combined application of transmission-and fluorescence projections of Neurobiotin labeled axotomized fibers confirms that recovery of normal size is restored by regenerated afferents. Remarkably, SLOTy images reveal that degeneration of olfactory receptor axons has a trans-synaptic effect on second order brain centers and leads to size reduction of the mushroom body calyx.

Conclusions

This study demonstrates that SLOTy is a suitable method for rapid screening of volumetric plasticity in insect brains and suggests its application also to vertebrate preparations.     

Oligodendrocyte N-Methyl-d-aspartate Receptor Signaling: Insights into Its Functions

Myelination by oligodendrocytes facilitates rapid nerve conduction. Loss of oligodendrocytes and failure of myelination lead to nerve degeneration and numerous demyelinating white matter diseases. N-methyl-d-aspartate (NMDA) receptors, which are key regulators on neuron survival and functions, have been recently identified to express in oligodendrocytes, especially in the myelin sheath. NMDA receptor signaling in oligodendrocytes plays crucial roles in energy metabolism and myelination. In the present review, we highlight the subcellular location-specific impairment of excessive NMDA receptor signaling on oligodendrocyte energy metabolism in soma and myelin, and the mechanisms including Ca²⁺ overload, acidotoxicity, mitochondria dysfunction, and impairment of respiratory chains. Conversely, physiological NMDA receptor signaling regulates differentiation and migration of oligodendrocytes. How can we use above knowledge to treat excitotoxic oligodendrocyte loss, congenital myelination deficiency, or postnatal demyelination? A thorough understanding of NMDA receptor signaling-mediated cellular events in oligodendrocytes at the pathophysiological level will no doubt aid in exploring effective therapeutic strategies for demyelinating white matter diseases.     

CD8+ T cells and IFN-γ mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria

Background

Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood.

Methods and Findings

Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8⁺ T cells and IFN-γ drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4⁺ T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-α did not influence the early increase of total parasite biomass and IRBC accumulation in different organs.

Conclusions

CD8⁺ T cells and IFN-γ are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues.     

Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

Introduction

Intervertebral disc (IVD) degeneration is characterized by extracellular matrix breakdown and is considered to be a primary cause of discogenic back pain. Although increases in pro-inflammatory cytokine levels within degenerating discs are associated with discogenic back pain, the mechanisms leading to their overproduction have not yet been elucidated. As fragmentation of matrix components occurs during IVD degeneration, we assessed the potential involvement of hyaluronic acid fragments (fHAs) in the induction of inflammatory and catabolic mediators.

Methods

Human IVD cells isolated from patient biopsies were stimulated with fHAs (6 to 12 disaccharides) and their effect on cytokine and matrix degrading enzyme production was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The involvement of specific cell surface receptors and signal transduction pathways in mediating the effects of fHAs was tested using small interfering RNA (siRNA) approaches and kinase inhibition assays.

Results

Treatment of IVD cells with fHAs significantly increased mRNA expression levels of interleukin (IL)-1β, IL-6, IL-8, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-1 and -13. The stimulatory effects of fHAs on IL-6 protein production were significantly impaired when added to IVD cells in combination with either Toll-like receptor (TLR)-2 siRNA or a TLR2 neutralizing antibody. Furthermore, the ability of fHAs to enhance IL-6 and MMP-3 protein production was found to be dependent on the mitogen-activated protein (MAP) kinase signaling pathway.

Conclusions

These findings suggest that fHAs may have the potential to mediate IVD degeneration and discogenic back pain through activation of the TLR2 signaling pathway in resident IVD cells.     

Delayed Nerve Stimulation Promotes Axon-Protective Neurofilament Phosphorylation,Accelerates Immune Cell Clearance and Enhances Remyelination In Vivo in Focally Demyelinated Nerves

Rapid and efficient axon remyelination aids in restoring strong electrochemical communication with end organs and in preventing axonal degeneration often observed in demyelinating neuropathies. The signals from axons that can trigger more effective remyelination in vivo are still being elucidated. Here we report the remarkable effect of delayed brief electrical nerve stimulation (ES; 1 hour @ 20 Hz 5 days post-demyelination) on ensuing reparative events in a focally demyelinated adult rat peripheral nerve. ES impacted many parameters underlying successful remyelination. It effected increased neurofilament expression and phosphorylation, both implicated in axon protection. ES increased expression of myelin basic protein (MBP) and promoted node of Ranvier re-organization, both of which coincided with the early reappearance of remyelinated axons, effects not observed at the same time points in non-stimulated demyelinated nerves. The improved ES-associated remyelination was accompanied by enhanced clearance of ED-1 positive macrophages and attenuation of glial fibrillary acidic protein expression in accompanying Schwann cells, suggesting a more rapid clearance of myelin debris and return of Schwann cells to a nonreactive myelinating state. These benefits of ES correlated with increased levels of brain derived neurotrophic factor (BDNF) in the acute demyelination zone, a key molecule in the initiation of the myelination program. In conclusion, the tremendous impact of delayed brief nerve stimulation on enhancement of the innate capacity of a focally demyelinated nerve to successfully remyelinate identifies manipulation of this axis as a novel therapeutic target for demyelinating pathologies.     

Anterograde Degeneration along the Visual Pathway after Optic Nerve Injury

Purpose

To investigate anterograde degenerative changes along the visual pathway in a rat model of optic nerve axotomy.

Methods

Optic nerve transection was performed in adult Sprague-Dawley rats. Animals were sacrificed at regular time intervals and tissues harvested. Immunoblotting followed by densitometric analysis was used to determine the phosphorylation profile of Akt in the dorsal lateral geniculate nucleus (dLGN) and the primary visual cortex (V1). The neuronal cell size and cell density were measured in the dLGN and the V1 using Nissl staining. The prevalence of apoptosis was characterized by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labelling (TUNEL) histochemistry. Caspase-3 antibodies were also used to identify apoptotic cells. Neurons and astrocytes were detected using NeuN and glial fibrillary acidic protein (GFAP), respectively.

Results

An early and sustained loss of Akt phosphorylation was observed after optic nerve transection in both dLGN and V1. At week one, a decrease in the neuronal cell size (50.5±4.9 vs 60.3±5.0 µm², P = 0.042) and an increase of TUNEL positive cells (7.9±0.6 vs 1.4±0.5 ×10² cells/mm², P<0.001) were evident in the dLGN but not in V1. A significant decline in neuronal cell number (14.5±0.1 vs 17.4±1.3 ×10² cells/mm², P = 0.048), cell size (42.5±4.3 vs 62.1±4.7 µm², P = 0.001) and an increase in apoptotic cells (5.6±0.5 vs 2.0±0.4 ×10² cells/mm², P<0.001) appeared in V1 initially at one month post-transection. The changes in the visual pathway continued through two months. Both neuronal cells and GFAP-positive glial cells were affected in this anterograde degeneration along the visual pathway.

Conclusions

Anterograde degeneration along the visual pathway takes place in target relay (LGN) and visual cortex following the optic nerve injury. Apoptosis was observed in both neural and adjacent glial cells. Reduction of Akt phosphorylation preceded cellular and apoptotic changes.     

The Low-Affinity Receptor for Neurotrophins p75NTR Plays a Key Role for Satellite Cell Function in Muscle Repair Acting via RhoA

Regeneration of muscle fibers, lost during pathological muscle degeneration or after injuries, is mediated by the production of new myofibres. This process, sustained by the resident stem cells of the muscle, the satellite cells, is finely regulated by local cues, in particular by cytokines and growth factors. Evidence in the literature suggests that nerve growth factor (NGF) is involved in muscle fiber regeneration; however, its role and mechanism of action were unclear. We have investigated this issue in in vivo mouse models of muscle regeneration and in primary myogenic cells. Our results demonstrate that NGF acts through its low-affinity receptor p75^NTR in a developmentally regulated signaling pathway necessary to myogenic differentiation and muscle repair in vivo. We also demonstrate that this action of NGF is mediated by the down-regulation of RhoA-GTP signaling in myogenic cells.     

Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae

D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S. cerevisiae, with or without co-expression of xylC (D-xylonolactonase), as seen by ¹H NMR. XylC facilitated rapid opening of the lactone and more D-xylonate was initially produced than in its absence. Using in vivo ¹H NMR analysis of cell extracts, culture media and intact cells we observed that the lactone and linear forms of D-xylonic acid were produced, accumulated intracellularly, and partially exported within 15–60 min of D-xylose provision.During single-cell analysis of cells expressing the pH sensitive fluorescent probe pHluorin, pHluorin fluorescence was gradually lost from the cells during D-xylonate production, as expected for cells with decreasing intracellular pH. However, in the presence of D-xylose, only 9% of cells expressing xylB lost pHluorin fluorescence within 4.5 h, whereas 99% of cells co-expressing xylB and xylC lost fluorescence, a large proportion of which also lost vitality, during this interval. Loss of vitality in the presence of D-xylose was correlated to the extracellular pH, but fluorescence was lost from xylB and xylC expressing cells regardless of the extracellular condition.     

Drosophila NMNAT maintains neural integrity independent of its NAD synthesis activity

Wallerian degeneration refers to a loss of the distal part of an axon after nerve injury. Wallerian degeneration slow (Wld^s) mice overexpress a chimeric protein containing the NAD synthase NMNAT (nicotinamide mononucleotide adenylyltransferase 1) and exhibit a delay in axonal degeneration. Currently, conflicting evidence raises questions as to whether NMNAT is the protecting factor and whether its enzymatic activity is required for such a possible function. Importantly, the link between nmnat and axon degeneration is at present solely based on overexpression studies of enzymatically active protein. Here we use the visual system of Drosophila as a model system to address these issues. We have isolated the first nmnat mutations in a multicellular organism in a forward genetic screen for synapse malfunction in Drosophila. Loss of nmnat causes a rapid and severe neurodegeneration that can be attenuated by blocking neuronal activity. Furthermore, in vivo neuronal expression of mutated nmnat shows that enzymatically inactive NMNAT protein retains strong neuroprotective effects and rescues the degeneration phenotype caused by loss of nmnat. Our data indicate an NAD-independent requirement of NMNAT for maintaining neuronal integrity that can be exploited to protect neurons from neuronal activity-induced degeneration by overexpression of the protein.     

Cystathionine β-synthase and cystathionine γ-lyase in tissues of the nemertean Cerebratulus marginatus Renier, 1804 (nemertea)

Using immunohistochemistry, we examined the distribution of the hydrogen sulfide (H₂S) synthesizing enzymes cystathionine β-synthase (CBS) and cystathionine Γ-lyase (CSE) in tissues of the nemertean Cerebratulus marginatus Renier, 1804 (Heteronemertea: Lineidae). The expression of both CBS and CSE was found in the body wall and in the receptor cells of the canals of the cerebral organs; the expression of CBS was found in the foregut and intestine; and CSE was found in the brain, lateral nerve cords, and cerebral organs. The peculiarities of the distribution and the possible functional role of H₂S-synthesizing elements in nemerteans are discussed.     

STRUCTURE OF THE MACULA UTRICULI WITH SPECIAL REFERENCE TO DIRECTIONAL INTERPLAY OF SENSORY RESPONSES AS REVEALED BY MORPHOLOGICAL POLARIZATION

The anatomy of the labyrinth and the structure of the macula utriculi of the teleost fish (burbot) Lota vulgaris was studied by dissection, phase contrast, and electron microscopy. The innervating nerve fibers end at the bottom of the sensory cells where two types of nerve endings are found, granulated and non-granulated. The ultrastructure and organization of the sensory hair bundles are described, and the finding that the receptor cells are morphologically polarized by the presence of an asymmetrically located kinocilium in the sensory hair bundle is discussed in terms of directional sensitivity. The pattern of orientation of the hair cells in the macula utriculi was determined, revealing a complicated morphological polarization of the sensory epithelium. The findings suggest that the interplay of sensory responses is intimately related to the directional sensitivity of the receptor cells as revealed by their morphological polarization. The problem of efferent innervation is discussed, and it is concluded that the positional information signaled by the nerve fibers innervating the vestibular organs comprises an intricate pattern of interacting afferent and efferent impulses     

LYSOSOMES IN THE RAT SCIATIC NERVE FOLLOWING CRUSH

Peripheral nerves undergoing degeneration are favorable material for studying the types, origins, and functions of lysosomes. The following lysosomes are described: (a) Autophagic vacuoles in altered Schwann cells. Within these vacuoles the myelin and much of the axoplasm which it encloses in the normal nerve are degraded (Wallerian degeneration). The delimiting membranes of the vacuoles apparently form from myelin lamellae. Considered as possible sources of their acid phosphatase are Golgi vesicles (primary lysosomes), lysosomes of the dense body type, and the endoplasmic reticulum which lies close to the vacuoles. (b) Membranous bodies that accumulate focally in myelinated fibers in a zone extending 2 to 3 mm distal to the crush. These appear to arise from the endoplasmic reticulum in which demonstrable acid phosphatase activity increases markedly within 2 hours after the nerve is crushed. (c) Autophagic vacuoles in the axoplasm of fibers proximal to the crush. The breakdown of organelles within these vacuoles may have significance for the reorganization of the axoplasm preparatory to regeneration. (d) Phagocytic vacuoles of altered Schwann cells. As myelin degeneration begins, some axoplasm is exposed. This is apparently engulfed by the filopodia of the Schwann cells, and degraded within the phagocytic vacuoles thus formed. (e) Multivesicular bodies in the axoplasm of myelinated fibers. These are generally seen near the nodes of Ranvier.