Effective gene transfer of lacZ and P0 into Schwann cells of P0-deficient mice.

Mutations in the gene encoding for the myelinating Schwann cell protein P0 have been linked to inherited peripheral neuropathies, including the Charcot-Marie-Tooth type 1B disease (CMT1B) and Dejerine-Sottas syndrome (DSS). Recently generated mice deficient in the P0 gene (P0-/- mice) resemble cases of CMT1B and DSS with impaired myelin dosage (Martini et al., 1995a). Potential approaches to treat such diseases include the introduction of the normal gene in the nerves of strongly affected patients. In the present study we used P0-/- mice to evaluate the efficiency of a replication-defective, E1-deleted adenovirus vector carrying the lacZ (Ad-RSV-lacZ) or P0 (Ad-RSV-P0) gene to infect abnormally myelinating Schwann cells. The Ad-RSV-lacZ vector suspension was injected into the left sciatic nerve ofPO-/- mice and the nerves examined for beta-galactosidase activity by X-gal histochemistry. Contralateral nerves injected with vehicle solution or non-injected served as controls. Beta-galactosidase activity was detected in nerves injected with the Ad-RSV-lacZ vector up to 2 weeks post-injection. Immunosuppressing the mice with FK506 to decrease the infiltration of activated T-cells in infected nerves lengthened beta-galactosidase activity to 8 weeks, the longest time point examined. Ultrastructural analysis indicated that X-gal crystals were present mostly in abnormally myelinating Schwann cells. These findings demonstrate that an adenovirus vector can successfully infect Schwann cells in P0-/- mice and expression can be maintained for several weeks. The Ad-RSV-P0 suspension was then injected in the sciatic nerve of immunosuppressed P0-/- mice. Two and four weeks post-injection both P0 mRNA and protein could be detected by in situ hybridization and Western blotting in some of the nerves. Furthermore, P0 protein expression was observed in myelin-like structures and onion bulb-like cells by immunohistochemistry. These results indicate that Schwann cells in P0-/- mice can be induced to produce P0 protein after gene transfer. Genetic repair of abnormal Schwann cells by using adenovirus vectors might be a possible technique to treat animal models of inherited peripheral neuropathies.

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.

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.

Onion bulb cells in mice deficient for myelin genes share molecular properties with immature, differentiated non-myelinating, and denervated Schwann cells.

Onion bulb formation is a pathological feature observed in peripheral nerves of patients suffering from inherited peripheral neuropathies such as Charcot-Marie-Tooth and Déjérine-Sottas diseases. An onion bulb consists of small circumferentially oriented (supernumerary) cells and their processes surrounding a large caliber axon. In the present study, we investigated the molecular phenotype of supernumerary cells at the light and electron microscopic levels. The major motor (quadriceps muscle) branch of the femoral nerve from 16- to 24-month-old mice with an inactivated allele of the myelin protein zero gene or deficient for myelin-associated glycoprotein (MAG; P0(+)- and MAG--mice, respectively), which have numerous onion bulbs, was teased to obtain single nerve fibers, which were then processed for immunocytochemistry. Corresponding nerves from wild-type mice served as controls. In both P0(+)- and MAG--mice, supernumerary cells expressed S-100, the low-affinity nerve growth factor receptor (p75, NGFr), the cell adhesion molecule L1, the neural cell adhesion molecule (N-CAM), and glial fibrillary acidic protein (GFAP). At the electron microscopic level, the cell surface of supernumerary cells was NGFr immunoreactive and L1 and N-CAM were expressed at points of contact between supernumerary cells. NGFr, L1, and N-CAM were also present in the basal lamina surrounding myelinated axons associated with onion bulbs. Both S-100 and GFAP immunoreactivities were seen in the cytoplasm of supernumerary cells. In contrast, in wild-type mice myelinating Schwann cells only expressed S-100 intracellularly and L1 and N-CAM in their basal lamina, whereas non-myelinating Schwann cells expressed all five molecules investigated. The present study indicates that supernumerary cells in onion bulbs have a molecular phenotype characteristic of immature, differentiated non-myelinating, and denervated Schwann cells, thus excluding the possibility that supernumerary cells are perineurial cells.

Effects of axonal injury on astrocyte proliferation and morphology in vitro: implications for astrogliosis.

Mechanisms inducing gliosis following injury in the central nervous sy stem are poorly understood. We evaluated the effect of axonal injury on astrocyte and Schwann cell proliferation and morphology in vitro. Purified rat dorsal root ganglion neurons grown on monolayers of rat neonatal cortical astrocytes (N-ASneonatal cultures) or sciatic nerve-derived Schwann cells (N-SC cultures) were mechanically injured. Non-injured cultures served as controls. Cell proliferation near lesions was monitored by autoradiography 1,2,4, and 8 days postinjury. Axonal injury caused a significant transient increase in astrocyte proliferation immediately proximal and distal to the lesion. The lesion did not induce marked changes in the intensity of glial fibrillary acidic protein (GFAP) immunoreactivity. However, processes from GFAP-positive cells usually arranged in random fashion in noninjured cultures were aligned perpendicularly to the cut distal to lesions. Ultrastructural analysis in lesioned N-ASneonatal cultures indicated that proximal to the lesion filament-filled astrocytes were intermingled with axons. Distal to the lesion astrocyte processes formed layers, between which an increased amount of collagen-like material appeared with time postlesion. Axons distal to the lesion degenerated by 2 days, coinciding with the early disappearance of neurofilament immunoreactivity. In noninjured and proximally in injured N-SC cultures, Schwann cells extended processes, engulfing some axons. Distal to the lesion, Schwann cells appeared more rounded and neurites remained until 4 days postinjury. Media conditioned by injured or non-injured N-ASneonatal cultures did not affect neuron-induced Schwann cell proliferation. These findings demonstrate that axonal injury and degeneration cause a transient increase in astrocyte proliferation and induce morphological changes in astrocytes consistent with the onset of gliosis.

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.

Effect of transforming growth factor-beta 1 and -beta 2 on Schwann cell proliferation on neurites.

Mechanisms regulating Schwann cell proliferation during development are unclear. Schwann cell division is known to be driven by an unidentified mitogen present on the surface of axons, but it is not known whether other molecules play a role in regulating this proliferation. Transforming growth factor-beta (TGF-beta) which is found in the developing peripheral nervous system (PNS) and is mitogenic for neuron-free Schwann cells in vitro could be involved. We have investigated the effects of TGF-beta 1, TGF-beta 2 and antibodies to TGF-beta 1 and TGF-beta 2 on axon driven Schwann cell proliferation. Rat embryonic dorsal root ganglion neurons (DRG) neurons and Schwann cells from the sciatic nerve were isolated, purified and recombined in vitro. Confirming earlier reports by others, we observed that TGF-beta 1 and TGF-beta 2 added to the culture medium stimulated the proliferation of Schwann cells in the absence of neurons. However, when added to neuron-Schwann cell co-cultures, TGF beta caused a variable response ranging from no effect to moderate inhibition of Schwann cell proliferation in different experiments. A stimulation of Schwann cell proliferation by TGF beta was never observed in neuron-Schwann cell co-cultures. Antibodies to TGF-beta 1 and TGF-beta 2 did not influence axon driven Schwann cell proliferation. To further determine the role of TGF-beta in Schwann cell proliferation and myelination, we studied Schwann cell proliferation in cultures from mice in which the TGF-beta 1 gene was delected by homologous recombination. Neuron-Schwann cell cultures from wild-type, heterozygous and homozygous mice were used. No differences were observed in either Schwann cell proliferation or myelination between cultures obtained from homozygous mutants and their heterozygous and wild-type controls. These findings suggest that TGF-beta does not function as a part of the mitogenic mechanism presented by neurons to Schwann cells, but that the presence of active TGF beta in the cellular environment might regulate the degree of proliferation induced by neuronal contact.

Transforming growth factor-beta blocks myelination but not ensheathment of axons by Schwann cells in vitro.

Mechanisms regulating Schwann cell differentiation into a myelinating or a mature nonmyelinating phenotype during development are poorly understood. Humoral factors such as members of the transforming growth factor-beta (TGF-beta) family, which are found in the developing and adult mammalian nervous system and are known to affect cell differentiation, could be involved. We tested the effects of TGF-beta isoforms on the ensheathment and myelination of dorsal root ganglion (DRG) neurons by Schwann cells in vitro. Rat embryonic DRG neurons and Schwann cells from the sciatic nerve were isolated, purified, and recombined. In serum-free conditions, TGF-beta blocked both Schwann cell myelination and the expression of the myelin-related molecules galactocerebroside, P0, myelin-associated glycoprotein, and myelin basic protein. In contrast, the expression of molecules characteristic of mature nonmyelinating Schwann cells, including neural-cell adhesion molecule, L1, and nerve growth factor receptor, was maintained when compared to Schwann cells in nondifferentiated cultures. Notably, the expression of glial fibrillary acidic protein, which is expressed only in mature nonmyelinating Schwann cells in vivo, was increased 10-fold in our cultures by TGF-beta. Electron microscopic analysis indicated that in the presence of TGF-beta, basal lamina deposition by Schwann cells was slightly increased. Most importantly, many axons in TGF-beta-treated cultures received ensheathment typical of mature nonmyelinated nerves. These effects of TGF-beta were partially reversed by specific neutralizing anti-TGF-beta antibodies. We interpret these results as evidence that TGF-beta regulates Schwann cell differentiation in vitro by blocking the expression of the myelinating phenotype and promoting the development of the nonmyelinating phenotype.

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.

Astrocytes inhibit Schwann cell proliferation and myelination of dorsal root ganglion neurons in vitro.

Schwann cells promote the regrowth of nerve fibers in both the PNS and CNS and might thus be of value in strategies to promote repair following injury or demyelination in the CNS. The effectiveness of Schwann cells in promoting repair could, however, be limited by interactions with reactive astrocytes that are prominent at lesioned and demyelinated sites. To investigate this possibility, experiments were performed to determine the influence of cortical astrocytes on Schwann cell proliferation and myelination of dorsal root ganglion (DRG) neurons in vitro. DRG neurons from embryonic rats and Schwann cells, astrocytes, and fibroblasts isolated from the sciatic nerve, cerebral cortex, and cranial periosteum, respectively, of neonatal rats were purified and then recombined to provide neuron-Schwann cell, neuron-Schwann cell-astrocyte, and neuron-Schwann cell-fibroblast cultures. Astrocytes inhibited both neuron-dependent Schwann cell proliferation and the myelination of axons by Schwann cells. The expression of galactocerebroside, but not of the O4 antigen, was inhibited by astrocytes, suggesting that astrocytes blocked Schwann cell differentiation prior to the onset of myelination. Ultrastructural analysis of the cultures also indicated that both axonal ensheathment and the segregation of large axons into 1:1 relationships were decreased in the presence of astrocytes. Astrocytes did not affect the expression of the basal lamina components type IV collagen and laminin, and basal lamina formation assessed by electron microscopy was only slightly decreased. Some of these inhibitory effects appear to be mediated by diffusible factors since astrocyte-conditioned medium also reduced Schwann cell myelination. Fibroblasts or fibroblast-conditioned medium did not induce such inhibitory effects, indicating that the effects were astrocyte specific. We conclude that cortical astrocytes release a soluble factor(s) that inhibits specific aspects of neuron-Schwann cell interactions leading to myelination.

The functional characteristics of Schwann cells cultured from human peripheral nerve after transplantation into a gap within the rat sciatic nerve.

The use of human Schwann cells (SCs) in transplantation to promote regeneration in central and peripheral neural tissues must be preceded by efforts to define the factors that regulate their functional expression. Adult-derived human SCs can be isolated and purified in culture, but the culture conditions that allow their full differentiation have not yet been defined. We tested the functional capacity of these cells to enhance axonal regeneration and myelinate regenerating axons in vivo by transplanting them into the damaged PNS of an immune-deficient rat. SCs were purified from human peripheral nerve obtained from organ donors. Semi-permeable guidance channels were filled with a 30% Matrigel containing solution with or without human SCs suspended at a density of 80 x 10(6) cells/ml. Channels were implanted within an 8 mm gap of the transfected sciatic nerve of nude female rats for a period of 4 weeks. Survival of the transplanted human SCs was established by dissociating nerve explants taken from the regenerated cable (after first placing them in culture for 5 d) and staining individual cells for a primate-specific NGF receptor (PNGFr) and S 100. Only one-half of the S 100-positive cells stained for the PNGFr, which indicated that the regenerated cable contained an approximately equal number of human and rat (host) SCs. The presence of some human myelin segments was confirmed by immune staining with an HNK-1 antibody that specifically labels human but not rat myelin. The majority of the myelin segments in the regenerated cable, however, were produced by the rat SCs. The number of myelinated axons and the cross-sectional area of the cable were significantly greater in channels seeded with human SCs when compared to channels containing the diluted Matrigel solution alone. We conclude that purified cultured human SCs can survive and substantially enhance axonal regeneration when transplanted into the injured PNS of an immune-deficient rat. Some of the transplanted human SCs are capable of myelinating regenerating rat axons but are less successful than the host SCs.

The astrocyte inhibition of peripheral nerve regeneration is reversed by Schwann cells.

Schwann cell transplantation into the lesioned or demyelinated central nervous system (CNS) is being extensively explored as an approach to favorably influencing repair in the CNS. Under a variety of circumstances, however, the CNS glial microenvironment appears to offer an unfavorable terrain for the promotion of neurite elongation and for Schwann cell differentiation. Due to the heterogeneity of the cellular contents at injury sites, the specific role of each cell type present in limiting Schwann cell function is unclear. The damaged peripheral nervous system, a system capable of substantial regeneration (and free of the potentially negative influence of oligodendrocytes), represents a valuable model in which to specifically evaluate the influence of astrocytes on Schwann cell function. In the present study, purified cortical astrocyte populations were seeded into semipermeable guidance channels alone or in combination with adult Schwann cell populations to determine their effects on regeneration across an 8-mm gap in the transected sciatic nerve of the adult rat. Channels were prepared with (or without) a defined cellular content, implanted in inbred Lewis rats and evaluated after 3 weeks. Channels seeded with astrocytes alone impeded regeneration, regardless of the maturity of the astrocytes (7-8 days vs 28 days in culture) and their seeding density (40 vs 80 x 10(6) cells/ml). On the other hand, Schwann cells derived from adult sciatic nerve seeded at similar densities enhanced the regenerative process. Regenerative capacity was diminished when astrocytes were combined with Schwann cells; the rate of regeneration increased as the number of Schwann cells in the astrocyte/Schwann cell mixture increased. Immunostaining of the nerve stumps related to astrocyte-seeded channels and of the regenerated tissue in the astrocyte-Schwann cell-seeded channels indicated that astrocytes had migrated into the proximal nerve stump; only a few astrocytes remained within the regenerated cable. The present experiments show that although astrocytes alone inhibit nerve regeneration, Schwann cells are able to partially overcome this inhibition if they are provided in sufficient numbers. We believe these observations will be valuable in considering clinical strategies to use autologous Schwann cell transplantation to influence CNS regeneration.

Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration.

At present, clinical strategies to repair injured peripheral nerve concentrate on efforts to attain primary suture of the cut nerve ends. If this is not possible, autografts are used to unite the separated nerve segments. Both strategies are based on the recognition that the Schwann cells resident in the peripheral nerve trunk play a crucial role in the regenerative process. Neither strategy may be feasible, however, in extensive or multiple injuries because the amount of autograft material is limited, and allografts are subject to immune rejection. Artificially produced nerve bridges constructed of autologous Schwann cells seeded in guidance channels could be used to overcome these limitations. In the present experiments, the potential of Schwann cells derived from adult nerves and seeded in permselective guidance channels to promote neurite regeneration across an 8 mm nerve gap was evaluated in transected rat sciatic nerves. Immunological sequalae were evaluated by comparing Schwann cells from syngeneic and heterologous rat strains. Schwann cells from either adult outbred (Sprague-Dawley, CD) rats or inbred (Fisher, F) rats were suspended in a Matrigel solution at a density of 80 x 10(6) cells/ml (CD) or 40, 80, or 120 x 10(6) cells/ml (F-40, F-80, and F-120 channels, respectively). Channels containing Schwann cells were compared to sciatic nerve autografts, empty channels, or channels filled with Matrigel alone. One day after seeding permselective synthetic guidance channels with a Schwann cell suspension, a central cable of Schwann cells oriented along the axis of the tube was formed due to syneresis of the hydrogel. By 3 weeks postimplantation, regenerating axons had grown into all channels and autografts. Sciatic nerve autografts supported extensive regeneration, containing 4-5 x 10(4) myelinated axons at the graft midpoint. The ability of channels containing syngeneic Schwann cells to foster regeneration was dependent on the Schwann cell seeding density. At the channel's midpoint, the myelinated axon population in F-120 tubes was intermediate between that in sciatic nerve autografts and F-80 channels, and was significantly higher than in F-40 or control channels. The nerve cable in Schwann cell-containing tubes consisted of larger, more organotypic fascicles than acellular control channels. In contrast, heterologous (CD) Schwann cells elicited a strong immune reaction that impeded nerve regeneration. The present study shows that cultured adult syngeneic Schwann cells seeded in permselective synthetic guidance channels support extensive peripheral nerve regeneration.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of dorsal and ventral spinal root regeneration through semipermeable guidance channels.

Semipermeable guidance channels have been shown to support nerve regeneration in the peripheral nervous system (PNS) possibly through interactions with the wound healing environment. This study quantitatively assesses the ability of such channels to support regeneration in the PNS segment of the spinal roots across a 4 mm gap and compares the resultant dorsal and ventral root regeneration. Acrylic copolymer guidance channels with a molecular weight (Mw) cutoff of 50,000 Da were used in a transected rat spinal root model. Cohorts of 23 animals (11 ventral, 12 dorsal) were examined at four weeks; 6 animals (3 ventral, 3 dorsal) at ten weeks; and 10 animals (5 ventral, 5 dorsal) at twenty-four weeks post-implantation. Both the dorsal and ventral roots were able to regenerate across the gap within the semipermeable channel. At all time periods, the regenerated dorsal roots contained fewer myelinated axons than found in the contralateral control root and consisted of an abundance of collagenous tissue. In contrast, by ten weeks the regenerated ventral roots contained twice the contralateral control number of myelinated axons and were composed predominantly of large, myelinated axons. At twenty-four weeks the number of unmyelinated axons was also quantified, with the regenerated dorsal root containing only one-fifth of the control number and the regenerated ventral root containing more than four times the control. Due to the proximity of the dorsal root lesion to the axonal cell bodies, the dorsal root ganglion (DRG) neuronal cell loss was investigated at four weeks post-implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral nerve regeneration is impeded by interleukin-1 receptor antagonist released from a polymeric guidance channel.

Interleukin-1 receptor antagonist (IL-1ra), a true antagonist of the interleukin-1 (IL-1) receptors, is released by activated macrophages and binds specifically to the IL-1 receptors without triggering IL-1 effects. Following peripheral nerve axotomy, activated macrophages release IL-1, which induces the expression of nerve growth factor (NGF) mRNA in Schwann cells. IL-1ra may therefore impede peripheral nerve regeneration by blocking the NGF-mediated effect of IL-1. Peripheral nerve regeneration occurring through polymeric guidance channels releasing IL-1ra was investigated in a 4-mm gap transected mouse sciatic nerve model. Cohorts of five animals were implanted with tubes releasing either bovine serum albumin (BSA), BSA with IL-1ra, or BSA with deactivated IL-1ra (dIL-1ra) for 4 weeks. In vitro release kinetics indicated that after an initial burst, IL-1ra release was linear for the next 3 1/2 weeks. Following implantation of a polymeric guidance channel, a regenerated cable bridged the nerve gap in all animals. The cables were similar in size and were composed of nerve microfascicles containing both unmyelinated and myelinated axons in association with their Schwann cells. Tissue regenerated in tubes releasing BSA-IL-1ra contained, however, significantly fewer myelinated and unmyelinated axons and blood vessels than did tubes releasing BSA alone or BSA-dIL-1ra. We conclude that a naturally occurring antagonist of IL-1 receptors impedes peripheral nerve regeneration, suggesting that macrophages play an essential role in controlling peripheral nerve regeneration through the release of stimulatory and/or inhibitory molecules.

Influence of surface texture of polymeric sheets on peripheral nerve regeneration in a two-compartment guidance system.

Synthetic guidance channels are useful tools to study the mechanisms underlying peripheral nerve regeneration. In the present study, the lumen of silicone elastomer tubes was divided into two compartments by a polymer strip 10 mm long placed along the tube length. The influence of varying the surface texture of hydrophilic and hydrophobic polymer strips on the morphology of the regenerated neural tissue was analysed. Hydrophilic nitrocellulose (NC) and hydrophobic polyvinylidene fluoride (PVDF) films with smooth (S-NC and S-PVDF) or rough (R-NC, R-PVDF) surface texture were used. Five channels of each type were used to repair transected rat sciatic nerves and analysed after 4 wk. Tissue strips bridged the nerve stumps in all R-NC and R-PVDF tubes, in five of the S-NC and three of the S-PVDF tubes. In R-NC and R-PVDF tubes, bell-shaped tissue adhering to the polymer strip was observed, whereas in S-NC and S-PVDF tubes round, free-floating nerve cables were seen. All the cables contained myelinated and unmyelinated axons and Schwann cells grouped in microfascicles and surrounded by an epineurial layer. For both rough strips, the initial cell layer consisted of macrophages adhering to the polymer surface. The epineurial nerve tissue contacting the rough surface was significantly thinner for PVDF compared with NC strips. No difference in epineurial thickness was observed for nerves facing the silicone tube or for smooth NC and PVDF strips. S-PVDF tubes contained significantly more myelinated axons than S-NC tubes.(ABSTRACT TRUNCATED AT 250 WORDS)

The morphology of regenerating peripheral nerves is modulated by the surface microgeometry of polymeric guidance channels.

The present study was designed to evaluate the influence of synthetic guidance channel surface microgeometry on morphological patterns of neural regeneration. Tubes with smooth (S), rough (R), or alternating smooth-rough (S/R) or rough-smooth (R/S) inner surfaces but with identical chemical composition and permeability characteristics were used to bridge a 4-mm nerve gap in a transected mouse sciatic nerve. Animals received S and R channels for 1, 2 and 4 weeks and both S/R and R/S channels for 2 and 4 weeks. At 1 week, the S tubes contained a longitudinally oriented fibrin matrix not contacting the channel's smooth inner wall, whereas R tubes featured an unorganized fibrin matrix which, together with fibroblasts and macrophages, had invaded the channel's rough trabecular network. After 4 weeks, S tubes contained discrete, free-floating nerve cables with numerous myelinated and unmyelinated axons surrounded by a thin, continuous epineurial-like layer, whereas R tubes were completely filled with a loose connective tissue stroma with only a few axons. In combined S/R or R/S channels, the general morphological patterns in individual S or R segments were similar to those observed in pure S or R channels, regardless of whether the tube segment was positioned at the proximal or distal nerve end. Proximal smooth channel segments contained discrete cables which abruptly fanned out to completely fill the lumen in distal rough segments. The opposite pattern was observed with proximal rough and distal smooth segments. At 4 weeks, myelinated axons were observed along the entire length of S/R and R/S tubes. These results suggest that the surface microgeometry of guidance channels influences the outcome of peripheral nerve regeneration, potentially by affecting the early arrangement of the fibrin matrix and/or inducing different cellular responses.

Peripheral nerve regeneration through blind-ended semipermeable guidance channels: effect of the molecular weight cutoff.

Synthetic nerve guidance channels are used to better understand the cellular and molecular events controlling peripheral nerve regeneration. In the present study, the contribution of wound-healing molecules to peripheral nerve regeneration was assessed by varying the molecular weight cutoff of the tubular membrane. Nerve regeneration through polysulfone tubular membranes with molecular weight (Mw) cutoffs of 10(5) and 10(6) Da was analyzed in a transected hamster sciatic nerve model. Cohorts of 6 animals received tubes of either type for 4 or 8 weeks with the distal end of the polymer tube capped. Other cohorts of 6 animals received tubes of either type for 4 weeks with the distal nerve stump secured within the guidance channel so as to create a 4 or 8 mm gap between both nerve stumps. Both types of channels contained regenerated tissue cables extending to the distal end of the guidance channel at both 4 and 8 weeks in the absence of a distal nerve stump. The cables regenerated in the 10(5) Da channels were composed of nerve fascicles surrounded by a loose epineurial sheath, whereas those regenerated in the 10(6) Da channels were composed mainly of granulation tissue. The numbers of myelinated and unmyelinated axons were significantly greater in the 10(5) Da than in the 10(6) Da channels at both 4 and 8 weeks. Both types of channel contained regenerated tissue cables with numerous nerve fascicles when the distal nerve stump was present with either gap length. However, when the gap distance was 8 mm, the 10(6) Da channels contained significantly fewer myelinated axons than the 10(5) Da channels. The present study reveals that the Mw cutoff of a semipermeable guidance channel strongly influences the outcome of peripheral nerve regeneration, possibly by controlling the exchange of molecules between the channel's lumen and the external wound-healing environment. These results suggest that the wound-healing environment secretes humoral factors that can either promote or inhibit the nerve-regeneration process.

VIP and acetylcholine: neurotransmitters in esophageal circular smooth muscle.

Vasoactive intestinal polypeptide (VIP) and acetylcholine were evaluated as possible inhibitory and excitatory neurotransmitters in the cat esophageal circular smooth muscle. Circular muscle strips 2 mm in thickness were obtained from 1 to 3.4 cm above the lower esophageal sphincter and tested in vitro. Muscle strips contracted with bethanechol (10(-5) M) were relaxed by electrical stimulation (0.5-5 Hz) and by VIP (10(-8)-10(-6) M). Relaxation induced by electrical stimulation was blocked by tetrodotoxin, whereas VIP-induced relaxation was not affected. Highly specific VIP antiserum (5%) antagonized both VIP and electrically induced relaxation, and the antagonism was eliminated when the antiserum was neutralized with VIP (10(-6.5) M). Dopamine (10(-4) M) reduced the relaxation induced both by exogenous VIP and by electrical stimulation but did not affect the relaxation caused by sodium nitroprusside (10(-8)-10(-5) M). In untreated strips, physostigmine (10(-10)-10(-8) M) enhanced the off contraction in response to electrical stimulation, whereas atropine caused a dose-dependent reduction with complete abolition at 10(-4) M. These data suggest that in the esophagus inhibition and excitation are mediated by distinct mechanisms: VIP mediates inhibition and acetylcholine is responsible for the off contraction in response to electrical stimulation.

Blind-ended semipermeable guidance channels support peripheral nerve regeneration in the absence of a distal nerve stump.

The presence of a distal nerve segment is considered to be essential for peripheral nerve regeneration through impermeable synthetic guidance channels. The use of a perm-selective material may provide a more appropriate regenerating environment by allowing solute exchange across the wall of the channel. We compared perm-selective acrylic copolymer (AC) channels with impermeable silicone elastomer (SE) channels in terms of regeneration in the absence of a distal nerve stump. Cohorts of 6 animals received AC and SE channels for either 4 or 8 weeks, with the distal end of the polymer tube left open in half of the animals, and plugged with the same polymer ('capped') in the other half. Capped and uncapped AC channels contained regenerated nerve cables which extended fully to the distal end of the channel, whereas capped SE channels contained only 1 mm long granulomatous tissue cables, and uncapped SE channels showed small cables with only a few myelinated axons. The nerve cables regenerated in uncapped AC channels were smaller and contained fewer myelinated axons than those observed in capped AC channels. Capped AC channels sleeved with a tight-fitting silicone tube to render them impermeable, showed no regenerated tissue within their lumen. The use of a perm-selective channel may have allowed the influx of nutrients and growth factors from the external environment while concentrating factors released by the proximal nerve stump.