Véronique Guénard

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.

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 8mm long at a density of 120×106 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 1mm 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 fibres 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.

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. GLIA 19:247–258, 1997.

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.

The use of schwann cell transplantation to foster central nervous system repair

Schwann cells (SCs) have been a key element in the demonstration of the ability of central nervous system (CNS) neurons to regenerate when provided with an appropriate environment. SCs transplanted into injured adult mammalian CNS (including the optic and septo-hippocampal systems, spinal cord and diencephalon) survive, promote axonal regeneration, and ensheathe or myelinate the regenerated axons. Methods to graft SCs into the lesioned CNS range from injection of a SC suspension to transplantation of more elaborate SC constructs using polymeric bridges. We discuss in detail the use of semipermeable guidance channels seeded with purified populations of SCs. These studies suggest that the ability of SCs to function in various injured CNS regions might lead to new avenues for the treatment of CNS injuries and demyelinating conditions in the human.

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.

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.