Axonal signals regulate expression of glia maturation factor-beta in Schwann cells: an immunohistochemical study of injured sciatic nerves and cultured Schwann cells

Glia maturation factor-beta (GMF-beta) is a 17 kDa protein purified and sequenced from bovine brains. Using the monoclonal antibody G2-09 directed against GMF-beta, we previously demonstrated endogenous GMF-beta in astroblasts, Schwann cells, and their tumors in culture. In the present study, we have used indirect immunofluorescence microscopy with G2-09 to examine the effects of transection, crush, and regeneration of sciatic nerve on the expression of GMF-beta in Schwann cells in situ and to study the time course of GMF-beta induction in Schwann cells in vitro. For comparison, a parallel study was carried out with monoclonal antibodies directed against nerve growth factor (NGF) receptor. We found that (1) neither GMF-beta nor NGF receptor was detectable in intact sciatic nerves, (2) all Schwann cells of the distal segment of the transected nerve expressed GMF-beta as early as 3 d after axotomy that persisted up to 3 weeks, (3) axonal regeneration repressed the Schwann cell expression of GMF-beta, (4) isolated Schwann cells derived from rat sciatic and adult human sural nerves developed intracellular GMF-beta in culture following an initial lag period, and (5) the induction of Schwann cell NGF receptor coincided temporally with that of GMF-beta in the transected nerve and in culture. These results show that the expression of GMF-beta in Schwann cells, as is the case with the NGF receptor, is induced by the loss of the normal axon-Schwann cell contact. We propose that the induction of GMF-beta, as well as NGF receptor, in Schwann cells after nerve injury plays a role in axonal regeneration.     

Postnatal development of rat peripheral nerves: an immunohistochemical study of membrane lipids common to non-myelin forming Schwann cells, myelin forming Schwann cells and oligodendrocytes

Interest in the role of membrane lipids in Schwann cell function prompted this study of lipid antigens on myelin- and non-myelin forming Schwann cells. Using the monoclonal antibodies 07, which recognises galactocerebroside, 08, 09 and 011, the distribution and time course of expression of the 4 membrane lipids have been determined in Schwann cells of the rat sciatic nerve and sympathetic trunk, derived from 1-60-day-old rats. The proportion of Schwann cells binding each monoclonal antibody was found by dissociating the nerves and allowing 3 h for the cells to attach to coverslips, prior to double label immunofluorescence, using the monoclonal antibody in conjunction with antibodies to S100 as a general Schwann cell marker, or P0 to distinguish cells which had formed myelin. All 4 lipid antigens were expressed by myelin forming Schwann cells, appearing just before, or at the time that the cells started to form myelin. Only 011 was restricted to myelin forming Schwann cells. Non-myelin forming Schwann cells expressed 07, 08 and 09. In the cervical sympathetic trunk, the developmental expression of these 3 lipids was essentially complete by postnatal day 20, whereas in the sciatic nerve, expression was not complete until days 40-60. The results show that the biochemical maturation in non-myelin forming Schwann cells differs greatly between different nerves, and may not be completed until several weeks postnatally. The results also demonstrate that in addition to galactocerebroside, other similarities exist in the lipid composition of myelin and the plasma membrane of non-myelin forming Schwann cells since the lipids defined by 08 and 09 antibodies are found among both Schwann cell variants.     

The fate of Schwann cell basement membranes in permanently transected nerves

After peripheral nerve fiber degeneration, Schwann cell basement membranes (SCBM) persist, maintain the columnar orientation of multiplying Schwann cells, and provide pathways for regenerating axons to original target tissue. Despite the putative importance of these functions, there is little quantitative information on SCBM in chronic denervation. The number, integrity, shape, and size of SCBM were evaluated in transverse electron micrographs of peroneal nerves of groups of mice at various times after permanent sciatic nerve transection. With increasing time after transection, the SCBM fragment, the fragments become shorter, dispersed throughout the endoneurium and partially disappear. The discontinuity, dispersion, and partial disappearance of SCBM, which worsens with time after nerve transection, alters the scaffold by which neurites grow back to formerly innervated or appropriate target tissue. Without reaching appropriate target tissue, regenerating fibers may fail to develop or undergo retrograde atrophy and degeneration. The progressive changes of the SCBM may contribute to the demonstrated poor regeneration when nerve reconnection is delayed, or when proximal nerves are reconnected, so that much time may elapse before neurites grow back to distal nerve.     

A double-transgenic mouse used to track migrating Schwann cells and regenerating axons following engraftment of injured nerves

We propose that double-transgenic thy1-CFP(23)/S100-GFP mice whose Schwann cells constitutively express green fluorescent protein (GFP) and axons express cyan fluorescent protein (CFP) can be used to serially evaluate the temporal relationship between nerve regeneration and Schwann cell migration through acellular nerve grafts. Thy1-CFP(23)/S100-GFP and S100-GFP mice received non-fluorescing cold preserved nerve allografts from immunologically disparate donors. In vivo fluorescent imaging of these grafts was then performed at multiple points. The transected sciatic nerve was reconstructed with a 1-cm nerve allograft harvested from a Balb-C mouse and acellularized via 7 weeks of cold preservation prior to transplantation. The presence of regenerated axons and migrating Schwann cells was confirmed with confocal and electron microscopy on fixed tissue. Schwann cells migrated into the acellular graft (163+/-15 intensity units) from both proximal and distal stumps, and bridged the whole graft within 10 days (388+/-107 intensity units in the central 4-6 mm segment). Nerve regeneration lagged behind Schwann cell migration with 5 or 6 axons imaged traversing the proximal 4 mm of the graft under confocal microcopy within 10 days, and up to 21 labeled axons crossing the distal coaptation site by 15 days. Corroborative electron and light microscopy 5 mm into the graft demonstrated relatively narrow diameter myelinated (431+/-31) and unmyelinated (64+/-9) axons by 28 but not 10 days. Live imaging of the double-transgenic thy1-CFP(23)/S100-GFP murine line enabled serial assessment of Schwann cell-axonal relationships in traumatic nerve injuries reconstructed with acellular nerve allografts.     

Axonal transport of putrescine, spermidine and spermine in normal and regenerating goldfish optic nerves

Radioactive putrescine, spermidine or spermine was injected into the right eye of normal goldfish and fish in which both optic nerves had been crushed 18 days earlier. Fish were sacrificed 0.25–21 days after injection. Trichloroacetic acid-soluble and -insoluble material was extracted from the right retina and both tecta and assayed for radioactivity (significant differences between left and right tecta suggesting axonal transport). The nature of the radioactivity in the TCA-soluble fraction was determined on an amino acid analyzer.

Results indicate that putrescine is not axonally transported in intact goldfish optic nerves, but that during regeneration of the optic nerve large amounts of putrescine are axonally transported at rates similar to the fast component of protein transport. Spermidine appears to be axonally transported both in intact optic nerves and in regenerating optic nerves, and at an intermediate rate of transport; the amount of spermidine transported is significantly increased during regeneration. Spermine is also axonally transported in intact and regenerating nerves, at a rate similar to the rapid rate of protein transport. The amount of spermine transported appears to be slightly less in regenerating than in intact nerves during early stages of regeneration, but increases during later stages of nerve regeneration.

The results suggest that putrescine and spermidine may be preferentially transported during nerve regeneration, while spermine and spermidine are transported extensively in intact nerves.     

Variable galactocerebroside expression by human Schwann cells in dissociated and peripheral nerve explant cultures

It has been well established that rat Schwann cells down regulate their cell-surface expression of galactocerebroside (GalC) in vitro under normal cell culture conditions. To determine whether human Schwann cells exhibit a similar down-regulation of GalC in vitro we examined GalC expression in dissociated human Schwann cell cultures derived from normal adult peripheral nerve. Twenty-four hours post-dissociation up to 63% of human Schwann cells were found to express detectable levels of GalC on their surface whereas less than 8% of the Schwann cells expressed detectable levels of GalC at 14 days post-dissociation. In contrast, after nearly 3 months of peripheral nerve explant culture, greater than 30% of human Schwann cells still retained their GalC expression. A similar pattern was also observed when analyzing Schwann cell purity with dissociated cultures exhibiting a rapid decrease in Schwann cell purity under normal culturing conditions although Schwann cell purity was found to be largely unaffected during the period of peripheral nerve explant culture. In summary, we found there was less variation in both GalC expression and Schwann cell purity with time in peripheral nerve explant cultures than dissociated cultures.     

Denervated skeletal muscle stimulates migration of Schwann cells from the distal stump of transected peripheral nerve: An in vivo study

We have tested the stimulation of Schwann cell migration from the distal stump of a 1 week transected sciatic nerve of adult rats by denervated skeletal muscle. Migrating Schwann cells were distinguished by the presence of non-specific cholinesterase (nChE) activity and glial fibrillary acidic protein (GFAP) at a distance of about 6 mm among denervated muscle fibres 4 weeks after insertion of the distal stump. In addition, the distal stump was introduced into the open end of a silicone chamber packed with artificial fibrin sponge (Gelaspon®) soaked in homogenate from intact or denervated muscles. A larger amount of migrated Schwann cells was observed in the chambers filled with homogenate from denervated muscles. An alteration in the amounts of Schwann cells migrating into the silicone chambers observed after histochemical staining (nChE or GFAP) was supported by biochemical measurements of the nChE activity. The biochemical assessment of the nChE activity revealed the increased amounts of migrated Schwann cells in proportion to the protein contents of homogenates from the denervated muscles. In addition, heating of homogenate from the denervated muscles resulted in a diminution of Schwann cell migration. Bromodeoxyuridine incorporation did not show an increased proliferation of Schwann cells inside the chambers following application of homogenate from the denervated muscles in comparison with the homogenate from the innervated muscles. Our results suggest a stimulation of Schwann cell migration from the distal stump of the transected sciatic nerve by soluble factor(s) produced by denervated skeletal muscles.     

Chemical communication between regenerating motor axons and Schwann cells in the growth pathway

There are receptors on denervated Schwann cells that may respond to the neurotransmitters that are released from growth cones of regenerating motor axons. In order to ascertain whether the interaction of the transmitters and their receptors plays a role during axon regeneration, we investigated whether pharmacological block of the interaction would reduce the number of motoneurons that regenerate their axons after nerve section and surgical repair. Peripheral nerves in the hindlimbs of rats and mice were cut and repaired, and various drugs were applied to the peripheral nerve stump either directly or via mini-osmotic pumps over a 2–4-week period to block the binding of acetylcholine to nicotinic and muscarinic acetylcholine receptors (AChRs: α-bungarotoxin, tubocurarine, atropine and, gallamine) and binding of ATP to P2Y receptors (suramin). In rats, the nicotinic AChR antagonistic drugs and suramin reduced the number of motoneurons that regenerated their axons through the distal nerve stump. In mice, suramin significantly reduced the upregulation of the carbohydrate HNK-1 on the Schwann cells in the distal nerve stump that normally occurs during motor axon regeneration. These data indicate that chemical communication between regenerating axons and Schwann cells during axon regeneration via released neurotransmitters and their receptors may play an important role in axon regeneration.     

Different effects of astrocytes and Schwann cells on regenerating retinal axons

Following a crush injury of the optic nerve in adult rats, the axons of retinal ganglion cells, stimulated to regenerate by a lens injury and growing within the optic nerve, are associated predominantly with astrocytes: they remain of small diameter (0.1-0.5 microm) and unmyelinated for > or = 2 months after the operation. In contrast, when the optic nerve is cut and a segment of a peripheral nerve is grafted to the ocular stump of the optic nerve, the regenerating retinal axons are associated predominantly with Schwann cells: they are of larger diameter than in the previous experiment and include unmyelinated axons (0.2-2.5 microm) and myelinated axons (mean diameter 2.3 microm). Thus, the grafted peripheral nerve, and presumably its Schwann cells, stimulate enlargement of the regenerating retinal axons leading to partial myelination, whereas the injured optic nerve itself, and presumably its astrocytes, does not. The result points to a marked difference of peripheral (Schwann cells) and central (astrocytes) glia in their effect on regenerating retinal axons.     

Oxidized galectin-1 stimulates the migration of Schwann cells from both proximal and distal stumps of transected nerves and promotes axonal regeneration after peripheral nerve injury

Oxidized galectin-1 has recently been identified as a key factor that plays important roles in initial axonal growth in injured peripheral nerves. The aim of this study was to investigate the effects of oxidized galectin-1 on regeneration of rat spinal nerves using acellular autografts (containing no viable cells) and allografts (containing no cell membranes) with special attention to the relationship between axonal regeneration and Schwann cell migration. Immunohistochemically, endogenous galectin-1 was expressed in dorsal root ganglion (DRG) neurons, spinal cord motoneurons, and axons and Schwann cells in normal sciatic nerves. Administration of oxidized recombinant human galectin-1 (rh-gal-lox, 5 ng/ml) in autograft model promoted axonal regeneration from motoneurons as well as from DRG neurons; this was confirmed by a fluorogold tracer study (p < 0.05). Anti-rh-gal-1 antibody (30 microg/ml) strongly inhibited axonal regrowth (p < 0.05). Pretreatment of allografts with rh-gal-lox stimulated the migration of Schwann cells not only from proximal stumps but also from distal stumps into the grafts, resulting in accelerated axonal regeneration (p < 0.05). Moreover, Schwann cell migration preceded the axonal growth in the presence of exogenous rh-gal-lox in the grafts. These results strongly suggest that local administration of exogenous rh-gal-lox promotes the migration of Schwann cells followed by axonal regeneration from both motor and sensory neurons, resulting in acceleration of neuronal repair. This technique may also be of value in the repair of human nerves.     

Axonal transport and localization of B-50/GAP-43-like immunoreactivity in regenerating sciatic and facial nerves of the rat

Neurons that can regenerate their axons following axotomy increase their synthesis and axonal transport of a growth-associated protein, called GAP-43, which has been shown to be identical to the synaptic phosphoprotein B-50. The function of B-50/GAP-43 to the process of regeneration is unknown. We used a polyclonal, affinity-purified antibody against B-50 to study the axonal transport and localization of B-50/GAP-43-like immunoreactivity (B50LI) in the regenerating sciatic and facial nerves of adult rats. Quantitative data were obtained by densitometry of the B-50 band in immunoblots of nerve segments, which had been run on SDS-polyacrylamide gels. In the regenerating sciatic nerve, anterograde accumulation at a collection ligature was 3.0 times higher than retrograde accumulation. The mobile fraction of B50LI was only 0.28 of total B50LI and traveled with a mean anterograde velocity of 5.3 mm/hr. B50LI distribution in the newly regenerated portion of the nerve revealed maximal B50LI levels midway between the position of the crush and the fastest-growing axons. Immunocytochemistry of this portion of the nerve demonstrated B50LI to be associated with regenerating axons but also to a large extent with extra-axonal structures outlining the Schwann cell bands of Büngner. This zone of B50LI-positive Schwann cell bands was found to extend more distally in nerves in which regeneration had processed longer, e.g., up to 5 mm distal to the crush after 3 d and 8 mm after 4 d. Further distal to this zone, many fine regenerating axonal profiles could be detected with B-50 antibody, but were neurofilament negative. These findings raise the possibility of an extra-axonal function of B-50/GAP-43, as this protein might be secreted from regenerating axons and might play a role in axon-Schwann cell interactions during axonal maturation.     

Multinucleated giant cells in AIDS encephalopathy: an immunohistochemical study

We report the neuropathological findings of 5 post-mortem cases of AIDS. The most common causes of the death were multiple opportunistic infections associated with cutaneous and/or visceral Kaposi sarcoma in two cases. Cerebral edema, demyelination and spongiosis of the white matter were present but th most remarkable finding was the presence of multinucleated giant cell (MGC) within and nearby microglial nodules. On immunohistochemical investigation MGC and microglial cells exhibit positive stain only for RCA I and Ferritin, while immunohistochemical markers for astrocytes, neurons, macrophages, histiocytes, lymphocytes and endothelial cells were negative. No microrganism, nor viral inclusions were detected. These results support the hypothesis that MGC may be derived from microglial cells.

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Sommario Gli AA. riportano gli aspetti neuropatologici di 5 casi autoptici in pazienti con AIDS la cui causa di morte era riferibile ad infezioni opportunistiche associate o meno a sarcoma di Kaposi cutaneo e/o viscerale. In tutti i casi era visibile un marcato edema cerebrale, demielinizzazione e spongiosi della sostanza bianca cerebrale, mentre gli aspetti caretteristici erano costituiti dalla presenza di cellule giganti multinucleate (CGM) poste in prossimità o all'interno dei noduli microgliali. Lo studio immunoistochimico ha dimostrato una netta positività cellulare per l'RCA I e per la Ferritina, mentre i markers per astrociti, neuroni, macrofagi, istiociti, linfociti e cellule endoteliali erano negativi. Questi risultati suffragano l'ipotesi che le CGM possono derivare da cellule microgliali.

     

Fine structural and immunohistochemical identification of perineurial cells connecting proximal and distal stumps of transected peripheral nerves at early stages of regeneration in silicone tubes

Perineurial cells are specialized connective tissue cells that form a barrier between endoneurium and epineurium in normal nerves. In the present study, the formation of the perineurium after transection of rat sciatic nerves was investigated. The cord bridging the gap between proximal and distal stumps through silicone tubes was studied 3, 7, 12, 18, and 21 days after surgery using electron microscopy and antibodies against epithelial membrane antigen (EMA), a marker for perineurial cells that has thus far not been applied to the study of differentiating cells in nerve tubulation systems. Initially, a thin cord consisting of fibrin bridged the gap between the stumps. At 7 days, longitudinal cells had migrated from both stumps toward the center of the tubes on the surface of the fibrin cord. These cells were immunoreactive with anti-EMA. At 12 days, ultrastructural features of perineurial cells (desmosomes, tight junctions, actin filaments with dense bodies, tonofilaments) were prominent in these cells. Subsequently, the gap was bridged through the perineurial tube by endothelial cells, pericytes, fibroblasts, Schwann cells, and axons. At 21 days, a single large nerve fascicle ensheathed by a mature perineurium was found between the stumps. Thus, the first cells to connect proximal and distal stumps in the investigated nerve regeneration silicon chamber system are perineurial cells. Through the tube formed by these cells, blood vessels and nerve fibers bridge the gap. Therefore, establishment of a perineurial connection between nerve stumps appears to be important in the sequence of events during nerve regeneration.The results of this study were presented in part at the Post Graduate Boerhaave Course: Brachial Plexus Injury, Leyden, March 25 and 26, 1993 [17] and at the 38th Annual Meeting of the Deutsche Gesellschaft für Neuropathologie and Neuroanatomie, Berlin, October 6–9, 1993 [27]     

Transplantation of olfactory ensheathing cells or Schwann cells restores rapid and secure conduction across the transected spinal cord

Olfactory ensheathing cells (OECs) or Schwann cells were transplanted into the transected dorsal columns of the rat spinal cord to induce axonal regeneration. Electrophysiological recordings were obtained in an isolated spinal cord preparation. Without transplantation of cells, no impulse conduction was observed across the transection site; but following cell transplantation, impulse conduction was observed for over a centimeter beyond the lesion. Cell labelling indicated that the regenerated axons were derived from the appropriate neuronal source, and that donor cells migrated into the denervated host tract. As reported in previous studies, the number of regenerated axons was limited. Conduction velocity measurements and morphology indicated that the regenerated axons were myelinated, but conducted faster and had larger axon areas than normal axons. These results indicate that the regenerated spinal cord axons induced by cell transplantation provide a quantitatively limited but rapidly conducting new pathway across the transection site.     

Axonal transport of the cytoskeleton in regenerating motor neurons: constancy and change

We have examined slow axonal transport in regenerating motor neurons of the rat sciatic nerve. Using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) we previously found that the slow component is the vehicle for the axonal cytoskeletal proteins, i.e. the neurofilament triplet proteins, tubulin and actin. When these proteins are pulse-labeled by injecting [3H]- or [35S]-amino acids into the spinal cord, they are transported distally in the nerve as two distinguishable waves of radioactivity, SCa and SCb. In normal motor neurons, the neurofilament triplet proteins and the tubulin are transported in SCa at an average velocity of 1.7 mm/day; the less heavily labeled SCb which moves at 2-5 mm/day is the primary vehicle for actin. We now find that during regeneration the velocity of SCa is unchanged in the region of the axon between the cell body and the lesion, but the amount of labeled neurofilament triplet and associated tubulin transported in the axon is decreased in neurons which had been labeled 20 days post-lesion. In contrast, the labeling of the slowly transported proteins moving ahead of the neurofilament triplet is greater in regenerating nerves than in controls. On the basis of our findings, we propose that in motor axons the normal supply of cytoskeletal protein, which is continuously transported in the slow component, is sufficient to support regeneration. Nevertheless, the neuron cell body can alter the supply of these cytoskeletal proteins so as to enhance its regenerative capacity.     

Nerve growth factor signaling of p75 induces differentiation and ceramide-mediated apoptosis in Schwann cells cultured from degenerating nerves.

In peripheral nerve regeneration or remyelination, immature Schwann cells expressing p75(NTR) play cardinal roles in the support and regeneration of axons (Griffin JW, Hoffman PN. Peripheral Neuropathy 361-376, 1993). Only one of four to six Schwann cells participate in remyelination of damaged or regenerating axons. The rest of the cells, or supernumerary Schwann cells, show severe atrophy and gradually decrease in number, reestablishing a 1:1 axon-Schwann cell relationship (Said G, Duckett S. Acta Neuropathol (Berl) 53:173-179, 1981). Recent reports demonstrated that severely atrophied supernumerary Schwann cells are eliminated by apoptosis during axonal regeneration or remyelination (Hirata H, Hibasami H. Apoptosis 3:353-360, 1998; Berciano MT, Calle E. Acta Neuropathol (Berl) 95:269-279, 1998). The mechanism to induce selective death of supernumerary Schwann cells without causing any damage to axon-associated Schwann cells or axons remains to be determined. In this article, we report that p75(NTR), the low-affinity receptor for all members of neurotrophins, signals both cell differentiation and apoptosis through intracellular ceramide elevation. The final response is dependent on the intracellular ceramide level and Schwann cells modulate their response by changing expression level of p75(NTR). This effect was selective for nerve growth factor (NGF). Taken together, the present study suggests that NGF contributes both to phenotypic regulation and to elimination of the dedifferentiated Schwann cells, while supporting survival or regeneration of certain types of axons during peripheral nerve repair or regeneration.     

Schwann cells depleted of galactocerebroside express myelin-associated glycoprotein and initiate but do not continue the process of myelination.

Two peripheral myelin components, galactocerebroside (GalC) and myelin-associated glycoprotein (MAG), are known to be expressed early in Schwann cell differentiation, prior to the formation of definitive myelin segments containing compacted membrane. To discern the relative roles of these myelin components, cultures of Schwann cells and dorsal root ganglion neurons were treated with antigalactocerebroside mAbs in order to remove GalC from the Schwann cell surface (Ranscht et al., 1987). In the continuous presence of anti-GalC antibodies and in a medium containing serum plus ascorbic acid, Schwann cells assemble a basal lamina and progress to the one:one stage of Schwann cell:axon interaction but do not differentiate further. Immunostaining with anti-MAG antibodies revealed that GalC-depleted Schwann cells expressed high levels of MAG. Double staining with anti-MAG and anti-P0 antibodies showed that there was essentially no P0 immunoreactivity in the same cells. In those Schwann cells that had attained a one:one association with large-diameter axons, the inner-axon-related cytoplasmic process had passed under the outer mesaxon but had not completed a full turn around the axon. The expression of MAG on the single cytoplasmic process apposed to the axon in Schwann cells depleted of GalC further implicates MAG in the initial envelopment of the axon during myelination.