PubMed Health⌕ Search

Biomedical subjects

M B Bunge

Publications and source records attributed to M B Bunge.

At least 37 records · Page 2Linked to original sources
PubMed1997

Actin plays a role in both changes in cell shape and gene-expression associated with Schwann cell myelination.

Schwann cell (SC) differentiation into a myelinating cell requires concurrent interactions with basal lamina and an axon destined for myelination. As SCs differentiate, they undergo progressive morphological changes and initiate myelin-specific gene expression. We find that disrupting actin polymerization with cytochalasin D (CD) inhibits myelination of SC/neuron co-cultures. Basal lamina is present, neurons are healthy, and the inhibition is reversible. Electron microscopic analysis reveals that actin plays a role at two stages of SC differentiation. At 0.75-1.0 microg/ml CD, SCs do not differentiate and appear as "rounded" cells in contact with axons. This morphology is consistent with disruption of actin filaments and cell shape changes. However, at 0.25 microg/ml CD, SCs partially differentiate; they elongate and segregate axons but generally fail to form one-to-one relationships and spiral around the axon. In situ hybridizations reveal that SCs in CD-treated cultures do not express mRNAs encoding the myelin-specific proteins 2',3'-cyclic nucleotide phosphodiesterase (CNP), myelin-associated glycoprotein (MAG), and P0. Our results suggest that at the lower CD dose, SCs commence differentiation as evidenced by changes in cell shape but are unable to elaborate myelin lamellae because of a lack of myelin-specific mRNAs. We propose that F-actin influences myelin-specific gene expression in SCs.

Actins↗
PubMed1997

A combination of insulin-like growth factor-I and platelet-derived growth factor enhances myelination but diminishes axonal regeneration into Schwann cell grafts in the adult rat spinal cord.

Insulin-like growth factor-I (IGF-I) promotes axonal regeneration in the peripheral nervous system and this effect is enhanced by platelet-derived growth factor (PDGF). We decided, therefore, to study the effects of these factors on axonal regeneration in the adult rat spinal cord. Semipermeable polymer tubes, closed at the distal end, containing Matrigel mixed with cultured rat Schwann cells and IGF-I/PDGF, were placed at the proximal stump of the spinal cord after removal of the thoracic T9-11 segments. Control animals received implants of only Matrigel and Schwann cells or only Matrigel and IGF-I/PDGF. Four weeks after implantation, electron microscopic analysis showed that the addition of IGF-I/PDGF resulted in an increase in the myelinated:unmyelinated fiber ratio from 1:7 to 1:3 at 3 mm in the Schwann cell graft, and that myelin sheath thickness was increased 2-fold. The reduced number of unmyelinated axons was striking in electron micrographs. These results suggested that IGF-I/PDGF enhanced myelin formation of regenerated axons in Schwann cell implants, but there was a 36% decrease in the total number of myelinated axons at the 3 mm level of the graft. This finding and the altered myelinated:unmyelinated fiber ratio revealed that the overall fiber regeneration into Schwann cell implants was diminished up to 63% by IGF-I/PDGF. Histological evaluation revealed that there were more larger cavities in tissue at the proximal spinal cord-graft interface in animals receiving a Schwann cell implant with IGF-I/PDGF. Such cavitation might have contributed to the reduction in axonal ingrowth. In sum, the results indicate that whereas the combination of IGF-I and PDGF enhances myelination of regenerating spinal cord axons entering implants of Matrigel and Schwann cells after midthoracic transection, the overall regeneration of axons into such Schwann cell grafts is diminished.

Animals↗
PubMed1997

The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord.

Advances in the purification and expansion of Schwann cells (SCs) from adult human peripheral nerve, together with biomaterials development, have made the construction of unique grafts with defined properties possible. We have utilized PAN/PVC guidance channels to form solid human SC grafts which can be transplanted either with or without the channel. We studied the ability of grafts placed with and without channels to support regeneration and to influence functional recovery; characteristics of the graft and host/graft interface were also compared. The T9-T10 spinal cord of nude rats was resected and a graft was placed across the gap; methylprednisolone was delivered acutely to decrease secondary injury. Channels minimized the immigration of connective tissue into grafts but contributed to some necrotic tissue loss, especially in the distal spinal cord. Grafts without channels contained more myelinated axons (x = 2129 +/- 785) vs (x = 1442 +/- 514) and were larger in cross-sectional area ( x = 1.53 +/- 0.24 mm2) vs (x = 0.95 +/- 0.86 mm2). The interfaces formed between the host spinal cord and the grafts placed without channels were highly interdigitated and resembled CNS-PNS transition zones; chondroitin sulfate proteoglycans was deposited there. Whereas several neuronal populations including propriospinal, sensory, motoneuronal, and brainstem neurons regenerated into human SC grafts, only propriospinal and sensory neurons were observed to reenter the host spinal cord. Using combinations of anterograde and retrograde tracers, we observed regeneration of propriospinal neurons up to 2.6 mm beyond grafts. We estimate that 1% of the fibers that enter grafts reenter the host spinal cord by 45 days after grafting. Following retrograde tracing from the distal spinal cord, more labeled neurons were unexpectedly found in the region of the dextran amine anterograde tracer injection site where a marked inflammatory reaction had occurred. Animals with bridging grafts obtained modestly higher scores during open field [(x = 8.2 +/- 0.35) vs (x = 6.8 +/- 0.42), P = 0.02] and inclined plane testing (x = 38.6 +/- 0. 542) vs (x = 36.3 +/- 0.53), P = 0.006] than animals with similar grafts in distally capped channels. In summary, this study showed that in the nude rat given methylprednisolone in combination with human SC grafts, some regenerative growth occurred beyond the graft and a modest improvement in function was observed.

Adolescent↗
PubMed1997

Bridging Schwann cell transplants promote axonal regeneration from both the rostral and caudal stumps of transected adult rat spinal cord.

Transplantation of cellular components of the permissive peripheral nerve environment in some types of spinal cord injury holds great promise to support regrowth of axons through the site of injury. In the present study, Schwann cell grafts were positioned between transected stumps of adult rat thoracic spinal cord to test their efficacy to serve as bridges for axonal regeneration. Schwann cells were purified in culture from adult rat sciatic nerve, suspended in Matrigel: DMEM (30:70), and drawn into polymeric guidance channels 8 mm long at a density of 120 x 10(6) cells ml-1. Adult Fischer rat spinal cords were transected at the T8 cord level and the next caudal segment was removed. Each cut stump was inserted 1 mm into the channel. One month later, a bridge between the severed stumps had been formed, as determined by the gross and histological appearance and the ingrowth of propriospinal axons from both stumps. Propriospinal neurons (mean, 1064 +/- 145 SEM) situated as far away as levels C3 and S4 were labelled by retrograde tracing with Fast Blue injected into the bridge. Near the bridge midpoint there was a mean of 1990 +/- 594 myelinated axons and eight times as many nonmyelinated, ensheathed axons. Essentially no myelinated or unmyelinated axons were observed in control Matrigel-only grafts. Brainstem neurons were not retrogradely labelled from the graft, consistent with growth of immunoreactive serotonergic and noradrenergic axons only a short distance into the rostral end of the graft, not far enough to reach the tracer placed at the graft midpoint. Anterograde tracing with PHA-L introduced rostral to the graft demonstrated that axons extended the length of the graft but essentially did not leave the graft. This study demonstrates that Schwann cell grafts serve as bridges that support (1) regrowth of both ascending and descending axons across a gap in the adult rat spinal cord and (2) limited regrowth of serotonergic and noradrenergic fibers from the rostral stump. Regrowth of monoaminergic fibres into grafts was not seen in an earlier study of similar grafts placed inside distally capped rather than open-ended channels. Additional intervention will be required to foster growth of the regenerated axons from the graft into the distal cord tissue.

Amidines↗
PubMed1996

Methylprednisolone administration improves axonal regeneration into Schwann cell grafts in transected adult rat thoracic spinal cord.

Schwann cell (SC) grafts support the regeneration of axons of numerous spinal cord neurons when placed into transected adult rat midthoracic spinal cord. Clinically, methylprednisolone (MP) has been shown to be neuroprotective if administered within 8 h after spinal cord injury. We investigated whether axonal regrowth into SC grafts is enhanced when MP is administered at the time of spinal cord transection and SC implantation. SCs from adult rat sciatic nerves were purified in culture, suspended in Matrigel, and drawn into semipermeable polymeric channels. MP (30 mg/kg) or vehicle (control) was administered intravenously at 5 min, 2 h, and 4 h to adult Fischer rats after transection at T8 and removal of the next three caudal segments. The rostral cord stump was inserted 1 mm into the channel; the distal end of the channel was capped. Thirty to forty-five days later, the SC/MP group showed large tissue cables in the channels and host cord tissue retained in the rostral end of the channels. Significantly more myelinated axons (1159 +/- 308) were present at the 5-mm level in SC/MP grafts (n = 6) than in SC/vehicle cables (355 +/- 108, n = 5). More unmyelinated than myelinated axons (approximately 4:1, n = 3) were resolved in the cables by electron microscopy. In the SC/MP group, unlike the SC/vehicle group, serotonergic and noradrenergic fibers were detected immunocytochemically 2.5 and 2.0 mm respectively, into the graft; astrocytes were also identified at similar distances from the interface. Fast Blue retrograde tracing (SC/MP, n = 4; SC/vehicle, n = 3) showed that more spinal cord neurons (1116 +/- 113 vs 284 +/- 88, respectively) and spinal cord neurons more distant from the graft (C8 vs C5) responded by extending axons into the graft in the presence of MP. Also, very significantly, supraspinal brain stem neurons extended axons into the graft only when MP was administered (mean 46 vs 0, n = 3). These results indicate that MP improves axonal regenerationn from both spinal cord and brain stem neurons into thoracic SC grafts, possibly by reducing secondary host tissue loss adjacent to the graft.

Animals↗
PubMed1996

Induction of mature neuronal properties in immortalized neuronal precursor cells following grafting into the neonatal CNS.

RN33B, a conditionally-immortalized neuronal cell line, survives and differentiates following grafting into the neocortex and hippocampus of adult and neonatal rat hosts. We have previously shown that these cells assume shapes characteristic of endogenous neurons at the integration site and persist up to 24 weeks post-grafting. In the present study we use electron microscopy and immunohistochemistry to characterize such cells. Differentiated RN33B cells were identical in size to endogenous neurons and their sizes depended on the specific location of integration. RN33B cells in the granule cell layer of the dentate gyrus and CA3 and CA1 pyramidal layers were 9.0, 15.3, and 12.6 microns in diameter, respectively. Grafted RN33B cells received synapses from fibres of host origin. Differentiated cells expressed neuronal markers, but not glial markers. Some differentiated cells expressed glutamate both in vitro and in vivo whereas undifferentiated cells did not. Grafted RN33B cells that differentiated with morphologies similar to CA3 pyramidal neurons and pyramidal cortical neurons expressed Py antigen, a neuronal marker that is differentially expressed in endogenous large pyramidal neurons of the cerebral cortex and large pyramids of hippocampal field CA3. This Py immunoreactivity was region-specific and corresponded to the endogenous pattern of Py immunostaining. Collectively, these data indicate that RN33B cells are capable of region-specific differentiation and have the potential to integrate functionally into the host CNS.

Animals↗
PubMed1995

Axonal regeneration into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord.

Schwann cells (SC) have been shown to promote regeneration in both the peripheral and central nervous systems. In this study we tested the ability of SC to enhance axonal regeneration in adult rat spinal cord by grafting SC-seeded guidance channels into transected cords. SC were purified in culture from adult inbred rat sciatic nerves, suspended in Matrigel, and seeded into semipermeable PAN/PVC channels (2.6 mm I.D. x 10 mm long) at a final density of 120 x 10(6) cells/ml. Channels filled with Matrigel alone served as controls. Adult isologous rat spinal cords were transected at the T8 level, and segments T9-T11 were removed. The rostral stump was inserted 1 mm into channels with capped distal ends. One month after grafting, a vascularized tissue cable was present within the channel in all animals. In SC-seeded channels (n = 14), a mean of 501 myelinated axons was found in the cable, and many axons extended 9-10 mm. Electron microscopy revealed typical SC ensheathment and myelination of axons with four times more unmyelinated than myelinated axons. Control channels (n = 8) contained fewer myelinated axons (mean = 71). When SC were prelabeled in culture with a nuclear dye, labeled nuclei were observed at 30 days, confirming SC survival. Astrocytes identified by glial fibrillary acidic protein staining did not migrate far into the cable, and prelabeled SC did not enter the cord. Lack of immunostaining for serotonin and dopamine beta-hydroxylase indicated that supraspinal axons did not regenerate into the cable. Fast Blue injections into the middle of the cable (n = 3) marked spinal cord interneurons (mean = 306) as far as nine segments rostral (25 mm, C7) extending axons into the graft; fewer dorsal root ganglion neurons were retrogradely labeled. In conclusion, purified populations of SC transplanted within channels promote both propriospinal and sensory axonal regeneration in the adult rat thoracic spinal cord.

Animals↗
PubMed1995

Addition of purified basal lamina molecules enables Schwann cell ensheathment of sympathetic neurites in culture.

Our aim was to determine the effect of purified basal lamina components on the differentiation of non-myelin-forming Schwann cells accompanying rat sympathetic neurons in culture. Previous work has demonstrated that Schwann cells contacting superior cervical ganglion neurons cultured in the presence of ascorbate and serum fail to effectively deposit matrix and ensheathe neurites unless fibroblasts are also present. We questioned if an increase in the amount of available basal lamina components could mimic fibroblast-induced Schwann cell differentiation. Superior cervical ganglion neuron plus Schwann cell cultures were supplemented with purified basal lamina molecules for up to 7 weeks. The addition of laminin, type IV collagen, heparan sulfate proteoglycan, or a combination of all three components, led to increased basal lamina deposition and increased neurite ensheathment compared with cultures with no additions. In long-term cultures receiving component additions, however, ensheathment was exaggerated, and the Schwann cells were hypertrophic. The ensheathing conformations adopted by these Schwann cells surpassed normal-appearing ensheathment and resembled more unusual unmyelinated nerve morphology found in vivo by others in normal senescence and in several peripheral neuropathies. These experiments show that increased availability of exogenous basal lamina components leads to increased basal lamina deposition and that basal lamina elaboration can have dramatic effects on the morphology of nonmyelinating Schwann cells. These findings suggest that influences of extracellular matrix should be considered when unusual Schwann cell/axon conformations are seen in vivo.

Animals↗
PubMed1995

A combination of BDNF and NT-3 promotes supraspinal axonal regeneration into Schwann cell grafts in adult rat thoracic spinal cord.

We previously demonstrated that Schwann cells (SCs) in semipermeable guidance channels promote axonal regeneration in adult rat spinal cord transected at the mid-thoracic level. Propriospinal but not supraspinal axons grew into these channels. Here, we tested the ability of exogenous brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) to promote axonal regeneration in this novel model. The two neurotrophins were delivered simultaneously into the channel by an Alzet minipump at a rate of 12 micrograms/day for each neurotrophin for 14 of 30 days tested; phosphate-buffered saline, the vehicle solution, was used as a control. Significantly more myelinated nerve fibers were present in SC/neurotrophin grafts than in SC/vehicle grafts (1523 +/- 292 vs 882 +/- 287). In the graft, at least 5 mm from the rostral cord-graft interface, some nerve fibers were immunoreactive for serotonin, a neurotransmitter specific to raphe-derived axons in rat spinal cord. Fast blue retrograde tracing from SC/neurotrophin grafts revealed labeled neurons in 10 nuclei of the brain stem, 67% of these being in the lateral and spinal vestibular nuclei. The mean number of labeled brain stem neurons in the SC/neurotrophin group (92; n = 3) contrasted with the mean in the SC/vehicle group (6; n = 4). Our results clearly demonstrate that BDNF and NT-3 infusion enhanced propriospinal axonal regeneration and, more significantly, promoted axonal regeneration of specific distant populations of brain stem neurons into grafts at the mid-thoracic level in adult rat spinal cord.

Animals↗
PubMed1995

Schwann cells degrade myelin and proliferate in the absence of macrophages: evidence from in vitro studies of Wallerian degeneration.

Interruption of axonal continuity in peripheral nerve trunks leads to axonal and myelin breakdown and removal distal to the injury site, a process known as Wallerian degeneration. Clearance of axonal and myelin debris has been attributed to the cooperative actions of two cell types, the indigenous Schwann cells and macrophages recruited to the regions of tissue damage. Recent work in this area has suggested a limited role for Schwann cells in myelin degradation and has emphasized the role of macrophages, not only in myelin clearance but also in the stimulation of Schwann cell proliferation which also occurs during Wallerian degeneration. In this report, we demonstrate that rat Schwann cells are capable of substantial myelin degradation unaided by macrophages. Observations were made following excision of neuronal somata from well-myelinated rat dorsal root ganglion neuron/Schwann cell co-cultures. The various stages of myelin breakdown were observed by phase microscopy, Sudan black staining, or electron microscopy. The time course for breakdown of individual myelin internodes varied from 2 to 10 days after injury and was to some extent dependent upon the original internodal length. Additionally, we show that most Schwann cells involved in Wallerian degeneration in the absence of macrophages undergo cell division following degradation of myelin into granules visible by light microscopy. The co-cultures employed were essentially free of macrophages as assessed by immunostaining for the OX42, ED2, and ED1 macrophage markers. No macrophages were detected by light or electron microscopy in the vicinity of the identified Schwann cells and furthermore, macrophages/monocytes were rarely observed in uninjured co-cultures as assessed by fluorochrome-conjugated acetylated LDL labelling. These results provide evidence in support of the ability of Schwann cells to carry out degradation of short myelin segments and to proliferate without macrophage assistance during Wallerian degeneration in vitro.

Animals↗
PubMed1994

Anti-beta 1 integrin antibody inhibits Schwann cell myelination.

Schwann cells (SCs) co-cultured with sensory neurons require ascorbate supplementation for basal lamina assembly and differentiation into myelinating cells. The ascorbate requirement can be bypassed by adding a purified basal lamina component, laminin, to SC/neuron co-cultures. We have examined the role of laminin receptors, namely, the beta 1 subfamily of integrins, in the process of myelination. We demonstrate by immunostaining or immunoprecipitation that undifferentiated SCs in contact with axons express large amounts of the beta 1 subunit in association with the alpha 1 or alpha 6 subunit. In co-cultures of myelinating SCs, alpha 1 beta 1 is no longer present, alpha 6 beta 1 is still present but at reduced levels, and alpha 6 beta 4 is expressed at much higher levels than in co-cultures of undifferentiated SCs. Immunogold labelling at the electron microscope level suggested that beta 1 integrins are randomly distributed on undifferentiated SCs, become localized to the SC surface contacting basal lamina in differentiating SCs before the onset of myelination, and are not detected on myelinating SCs. Fab fragments of beta 1 function-blocking antibody block both attachment of isolated SCs to laminin and formation of myelin sheaths by SCs co-cultured with neurons in ascorbate-supplemented medium. SCs unable to myelinate in the presence of the anti-beta 1 antibody assemble patchy basal lamina that is only loosely attached to the cell surface and in some cases appears to be detaching from the membrane. In contrast, an alpha 1 beta 1 function-blocking antibody only partially blocks attachment of isolated SCs to laminin but has no inhibitory effect on SC myelination. These results are consistent with the hypothesis that a member of the beta 1 subfamily of integrins other than alpha 1 beta 1 binds laminin present in basal lamina to the SC surface and transduces signals that are critical for initiation of SC differentiation into a myelinating cell.

Animals↗
PubMed1994

Characterization of photochemically induced spinal cord injury in the rat by light and electron microscopy.

This study characterized by light and electron microscopy 49 photochemically induced lesions in adult rat spinal cord at 16 time intervals from 2 days to 17 months after lesioning. Vascular thrombosis, resulting from an intravascular photochemical reaction induced by a rose bengal/laser beam interaction, led within a few days to an extensive area of tissue deterioration. This area, termed the "lesion cavity" in contrast to the "secondary cavity" observed later, was at least 6 mm long and, at the epicenter, extended across most of the spinal cord width and from the dorsal surface to a level near the central canal. The area of spared tissue, 43% of the spinal cord cross-section at 2 days, did not change significantly between 2 and 56 days. Large numbers of macrophages populated the degenerating area by 5 days. This necrotic area was surrounded by a thin peripheral rim of largely intact white matter dorsally and laterally except at the epicenter where the white matter degenerated dorsomedially. In these peripheral regions, demyelination and, by 14 days, remyelination by both oligodendrocytes and Schwann cells (SCs) were evident. By 28 days, far more SCs (and meningeal cells) had entered the dorsal spinal cord, typically at the epicenter where meningeal thickening was most striking, and had migrated farther into the lesion cavity. These SCs and the axons they myelinated remained prominent in dorsal regions for many months, particularly at the epicenter; the proportion of SC to oligodendrocyte myelin diminished away from the epicenter. By 8 weeks, the lesion cavity was considerably diminished in size and thereafter it contained scattered macrophages, SC-myelinated axons, and blood vessels, primarily medially owing to flattening into clefts bilaterally. The cavity was partly bordered by astrocytes whose surfaces toward the lesion cavity were highly irregular and coated with basal lamina. Bare axons, consistently seen by electron microscopy at 5 days to 6 months, were typically ensconced among astrocytes starting at 28 days. Also by this time large, smoothly contoured, empty secondary cavities appeared, usually rostral and caudal to the epicenter; they did not increase in size or number with time. From 28 days to 17 months postlesion they occurred in 68% of the lesioned spinal cords. The secondary cavity border was composed of cells thought to be astrocytes but, surprisingly, the luminal surface was smooth and lacked basal lamina, in contrast to the primary lesion cavity border. Thus, two types of cavities formed after photochemical lesioning. This lesioning technique may provide an appropriate milieu to better understand aspects of the vexing problem of post-traumatic syringomyelia in the human.

Animals↗
PubMed1994

Transplantation of purified populations of Schwann cells into lesioned adult rat spinal cord.

Both peripheral nerve and purified populations of Schwann cells promote axonal regeneration in the peripheral and central nervous systems. In order to assess whether Schwann cells can provide a bridge enabling regrowth of descending and ascending axons across an area of injury in adult spinal cord, Schwann cells enclosed within a collagen scroll were transplanted into lesions created photochemically. Numerous myelinated and unmyelinated axons were found throughout 28-90 day implants; Schwann cells myelinated or ensheathed the ingrowing axons normally. In contrast, acellular collagen grafts did not contain axons. Thus, Schwann cells stimulated abundant growth of axons into the grafts. In part to address the concern that the dense collagen layer acted as a barrier, we assessed transplantation of Schwann cells, inside semipermeable polyacrylonitrile/polyvinylcholoride (PAN/PVC) guidance channels, after transection of adult inbred rat spinal cords at T8 with removal of the the T9-11 segments. One month after grafting, a vascularized tissue cable was present with more myelinated and unmyelinated axons in the Schwann cell seeded channels than controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylic Resins↗
PubMed1994

Regrowth of axons in lesioned adult rat spinal cord: promotion by implants of cultured Schwann cells.

Highly purified populations of Schwann cells were grafted into lesioned adult rat spinal cord to determine if they promote axonal regeneration. Dorsal spinal cord lesions were created by a photochemical lesioning technique. Schwann cells derived from E16 rat dorsal root ganglia, either elongated and associated with their extracellular matrix or dissociated and without matrix, were rolled in polymerized collagen to form an implant 4-6 mm long which was grafted at 5 or 28 days after lesioning. No immunosuppression was used. Acellular collagen rolls served as controls. At 14, 28 and 90 days and 4 and 6 months after grafting, animals were analysed histologically with silver and Toluidine Blue stains and EM. The grafts often filled the lesion and the host borders they apposed exhibited only limited astrogliosis. By 14 days, bundles of unmyelinated and occasional thinly myelinated axons populated the periphery of Schwann cell implants. By 28 days and thereafter, numerous unmyelinated and myelinated axons were present in most grafts. Silver staining revealed sprouted axons at the implant border at 28 days and long bundles of axons within the implant at 90 days. Photographs of entire 1 micron plastic cross-sections of nine grafted areas were assembled into montages to count the number of myelinated axons at the graft midpoint; the number of myelinated axons ranged from 517-3214. Electron microscopy of implants showed typical Schwann cell ensheathment and myelination, increased myelin thickness by 90 days, and a preponderance of unmyelinated over myelinated axons. Random EM sampling of five Schwann cell grafts showed that the ratio of unmyelinated to myelinated axons was highest (20:1) at 28 days. These ratios implied that axons numbered in the thousands at the graft midpoint. Dissociated Schwann cells without matrix promoted axonal ingrowth and longitudinal orientation as effectively as did elongated Schwann cells accompanied by matrix. There was a suggestion that axonal ingrowth was at least as successful, if not more so, when the delay between lesioning and grafting was 28 rather than 5 days. Acellular collagen grafts did not contain axons at 28 days, the only interval assessed. In sum, grafts of Schwann cells in a rolled collagen layer filled the lesion and were well tolerated by the host. The Schwann cells stimulated rapid and abundant growth of axons into grafts and they ensheathed and myelinated these axons in the normal manner.

Animals↗
PubMed1993

Fibroblasts promote Schwann cell basal lamina deposition and elongation in the absence of neurons in culture.

We and other investigators have repeatedly observed that, although purified populations of Schwann cells cultured in the absence of neurons immunostain for some basal lamina constituents, assembled basal lamina is not seen ultrastructurally. In this study we show that purified rat Schwann cells cultured with either fibroblasts or diffusible substances from fibroblasts acquire basal lamina; this basal lamina is most robust and continuous when Schwann cells and fibroblasts are cultured together. The diffusible fibroblast factor(s) is ascorbate-dependent and heat-sensitive and exhibits a molecular weight greater than 30-50,000. The addition of either a combination of basal lamina components or purified laminin also results in the deposition of lengths of basal lamina. Extracellular matrix elaboration promoted by fibroblast factors is linked to dramatic changes in the morphology of Schwann cells; the cells become elongated and aligned with respect to each other. The addition of purified laminin partly mimics this effect. These changes may represent an early response of the Schwann cell to the acquisition of basal lamina preceding further differentiation in vivo. Our observations resemble those reported for other epithelial cell-fibroblast interactions and thus reaffirm the identity of the Schwann cell as an epithelial cell sensitive to the influence of mesenchymally derived factors. We propose that fibroblast stimulation of Schwann cell extracellular matrix deposition probably occurs in normal development with axons and in abnormal situations without axons, such as peripheral nerve injury and tumor formation.

Animals↗
PubMed1993

Fibroblasts are required for Schwann cell basal lamina deposition and ensheathment of unmyelinated sympathetic neurites in culture.

The ability to purify and recombine populations of peripheral neurons, Schwann cells and fibroblasts in tissue culture has enabled us to examine the contribution of fibroblasts to Schwann cell basal lamina assembly and ensheathment of unmyelinated rat superior cervical ganglion neurites in vitro. Purified perinatal superior cervical ganglion neurons were grown in culture either with Schwann cells or with Schwann cells plus fibroblasts derived from either superior cervical ganglion capsule or cranial periosteum. The cultures were maintained for 2-8 weeks on a collagen substratum in a medium known to promote Schwann cell differentiation (myelin, basal lamina formation) in the presence of dorsal root ganglion neurons. The extent of Schwann cell differentiation (ensheathment, basal lamina formation) in the presence of superior cervical ganglion neurons was evaluated in this study using electron microscopy. In superior cervical ganglion neuron plus Schwann cell cultures (without fibroblasts), Schwann cells achieved only a moderate degree of ensheathment; also, Schwann cell basal lamina was discontinuous and extracellular collagen fibrils were sparse. Although only discontinuous basal lamina was demonstrable by electron microscopy in these cultures, surprisingly, Schwann cell/neurite fascicles were uniformly immunostained for laminin, type IV collagen, and heparan sulfate proteoglycan. The addition of fibroblasts to superior cervical ganglion neuron plus Schwann cell cultures increased the deposition of basal lamina around the Schwann cell/neurite units, the number of collagen fibrils, and the extent of neurite ensheathment. We propose that the presence of basal lamina increases the Schwann cell's ability to ensheathe superior cervical ganglion neurites, possibly through an augmentation of specific extracellular matrix components or by increasing in some way the capacity of these components to become organized into basal lamina. We conclude that, unlike dorsal root ganglion neurons, superior cervical ganglion neurons are unable to stimulate full Schwann cell extracellular matrix expression with the result that these Schwann cells require the extraneuronal influence of fibroblasts to deposit basal lamina and attain their mature phenotype in culture.

Animals↗
PubMed1993

Expression of the protein zero myelin gene in axon-related Schwann cells is linked to basal lamina formation.

A Schwann cell has the potential to differentiate into either a myelinating or ensheathing cell depending upon signals received from the axon that it contacts. Studies focusing on the pathway leading to myelination demonstrated that Schwann cells must form a basal lamina in order to myelinate an axon. In this report, we describe studies that indicate that initiation of basal lamina synthesis is required for Schwann cells to distinguish between myelination-inducing axons and axons that do not induce myelination, and to respond by undergoing the appropriate genetic and cellular changes. We have used high resolution in situ hybridization, immunocytochemistry and electron microscopy to examine changes in gene expression and morphology of Schwann cells differentiating into myelin-forming cells in vitro. These experiments were carried out in dorsal root ganglion neuron/Schwann cell co-cultures maintained in either serum-free, serum-only or serum-plus-ascorbate-containing medium. We have made four novel observations that contribute significantly to our understanding of how basal lamina and myelination are linked. (1) The addition of ascorbate (in the presence of serum), which promotes basal lamina production, appears to induce expression of the protein zero gene encoding the major structural protein of myelin. Moreover, expression of protein zero mRNA and protein, and its insertion into myelin membranes, occurs only in the subset of Schwann cells contacting myelination-inducing axons. Schwann cells in contact with axons that do not induce myelination, or Schwann cells that have not established a unitary relationship with an axon, do not express protein zero mRNA although they produce basal lamina components. (2) In serum-free conditions, a majority of Schwann cells express protein zero mRNA and protein, but this change in gene expression is not associated with basal lamina formation or with elongation of the Schwann cell along the axon and elaboration of myelin. (3) In the presence of serum (and the absence of ascorbate), Schwann cells again fail to form basal lamina or elongate but no longer express protein zero mRNA or protein. (4) Myelin-associated glycoprotein and galactocerebroside, two additional myelin-specific components, can be expressed by Schwann cells under any of the three culture conditions. Therefore, we have demonstrated that axonal induction of protein zero gene expression in Schwann cells is subject to regulation by both serum- and ascorbate-dependent pathways and that not all myelin-specific proteins are regulated in the same manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗