Neonatal Rat Sciatic Nerve Research Articles

Short and long-term neuroprotective effects of cannabidiol after neonatal peripheral nerve axotomy

Neonatal rat sciatic nerve crush mimics obstetric axonotmesis, leading to extensive loss of motor and sensory neurons. The present study aimed to investigate the neuroprotective potential of cannabidiol (CBD) and the role of cannabinoid receptors after sciatic nerve crush in neonatal rats. For that, two-day-old Wistar rats were used, organized into the following experimental groups: sciatic nerve crush plus CBD treatment (CBD), crush plus vehicle treatment (VE), crush + CBD + AM251 treatment (AM251 – CB1 inverse agonist), crush + CBD + AM630 treatment (AM630 – CB2 antagonist). Spinal motoneuron survival was evaluated by Nissl staining of the lumbar spinal cord, 5- and 56-days following injury. CBD treatment enhanced neuronal survival by ~54 % both 5 days and 8 weeks after injury. However, AM251 and AM630 treatment decreased neuronal rescue by 30 % when compared to the CBD group, suggesting that CBD acts partially through such pathways. However, in the long term, only the CB1 blockade reverted CBD positive results. Synaptic preservation was evaluated by anti-synaptophysin immunolabeling. Five days after the lesion, CBD treatment preserved ~35 % of synapses in the ventral horn, and such effect was partially reversed by CB1 inactivation. Additionally, CBD treatment reduced astroglial reaction both at 5 days (39 %, compared to VE) and 8 weeks (31 %, compared to VE) after lesion. The microglial response was acutely reduced by 62 % after CBD treatment. Overall, the results herein show that CBD is neuroprotective, increasing neuronal survival and reducing glial reaction after neonatal axotomy. Such effects require CB1 and CB2 receptors to be effective, in turn influencing neuroprotection, glial reactivity, and functional recovery.

Single-cell analyses reveal the differentiation shifts of Schwann cells in neonatal rat sciatic nerves.

Schwann cells provide essential physical and chemical supportfor neurons and play critical roles in the peripheral nervous system. To acquire an enhanced understanding of the genetic characteristics of Schwann cells, we analyzed single-cell transcriptional profiling of Schwann cells in neonatal rat sciatic nerves, ordered the pseudotemporal states of Schwann cells, and determined the magnitude of RNA velocity vectors as well as cell cycle stages of Schwann cell subtypes. We discovered the cellular heterogeneity of Schwann cells in neonatal rat sciatic nerves, revealed the dynamic changes of Schwann cell subtypes, and pointed out the differentiation trajectory from Timp3- and Col5a3-expressing Schwann cell subtype 3 to other Schwann cell subtypes. The functional interpretation further indicated that subtype 3 Schwann cells display genetic signatures of DNA replication and the acquisition of mesenchymal traits. Our study presents a transcriptional summarization of the differentiation states of Schwann cell subtypes in neonatal rat sciatic nerves at single-cell resolution and may serve as a foundation for a deeper comprehension of the involvement of Schwann cells in the development and regeneration of peripheral nerves.

Cell populations in neonatal rat peripheral nerves identified by single-cell transcriptomics.

Peripheral nerves connect central nerves with target tissues and organs and execute vital signal transduction functions. Although sub-types of neurons have been defined, the heterogeneity of cell populations in peripheral nerves, especially Schwann cells, has not been well demonstrated. Here, we collected sciatic nerves (SN) and dorsal root ganglia (DRG) from neonatal (1-day old) rats and classified cell populations by high-coverage single-cell sequencing. A total of 10 types of cells, including endothelial cells, erythrocytes, fibroblasts, monocytic cells, neurons, neutrophils, pericytes, satellite cells, Schwann cells, and vascular smooth muscle cells, were identified by transcriptome-based cell typing. The comparisons of cells in neonatal rat SN and DRG revealed distinct atlas in different tissue localizations. Investigations of ligand-receptor interactions showed that there existed direct cell-cell communications between endothelial cells and fibroblasts in SN and among endothelial cells, fibroblasts, and vascular smooth muscle cells in DRG. Schwann cells in neonatal rats were further sub-grouped to four sub-types, including LOC100134871 and Hbb expressing Schwann cell sub-type 1, Cldn19 and Emid1 expressing Schwann cell sub-type 2, Timp3 and Col5a3 expressing Schwann cell sub-type 3, and Cenpf and Mki67 expressing Schwann cell sub-type 4. These Schwann cell sub-types exhibited distinct genetic features and functional enrichments. Collectively, our results illustrated the diversity and cellular complexity of peripheral nerves at the neonatal stage and revealed the heterogeneity of Schwann cells in the peripheral nervous system.

Isolation and Expansion of Schwann Cells from Transgenic Mouse Models.

The most widely used method (Brockes' method) for preparing primary Schwann cell culture uses neonatal rat sciatic nerves as the primary source of Schwann cells. The procedure is relatively simple and yields a highly purified population of Schwann cells in a short period of time. The method has also been used to prepare Schwann cells from mice, however, with limitation. For example, Brockes' method is not applicable when the genotypes of mouse neonates are unknown or if the mouse mutants do not develop to term. We described a method ideal for preparing Schwann cells in a transgenic/knockout mouse study. The method uses embryonic dorsal root ganglia as the primary source of Schwann cells and allows preparing separate, highly purified Schwann cell cultures from individual mouse embryos in less than 2weeks.

MiR-30c promotes Schwann cell remyelination following peripheral nerve injury.

Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination following peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.

Peripheral nerve repair of transplanted undifferentiated adipose tissue‐derived stem cells in a biodegradable reinforced nerve conduit

This study proposes a biodegradable nerve conduit containing genipin-cross-linked gelatin annexed with tricalcium phosphate ceramic particles (genipin-gelatin-tricalcium phosphate, GGT) in peripheral nerve regeneration. Firstly, cytotoxicity tests revealed that the GGT-extracts were not toxic, and promoted the proliferation and neuronal differentiation of adipose tissue-derived stem cells (ADSCs). Secondly, the GGT composite film effectively supported ADSCs attachment and growth. Additionally, the GGT substrate was biocompatible with the neonatal rat sciatic nerve and produced a beneficial effect on peripheral nerve repair through in vitro tissue culture. Finally, the experiments in this study confirmed the effectiveness of a GGT/ADSCs nerve conduit as a guidance channel for repairing a 10-mm gap in a rat sciatic nerve. Eight weeks after implantation, the mean recovery index of compound muscle action potentials (CMAPs) was significantly different between the GGT/ADSCs and autografts groups (p < 0.05), both of which were significantly superior to the GGT group (p < 0.05). Furthermore, walking track analysis also showed a significantly higher sciatic function index (SFI) score (p < 0.05) and better toe spreading development in the GGT/ADSCs group than in the autograft group. Histological observations and immunohistochemistry revealed that the morphology and distribution patterns of nerve fibers in the GGT/ADSCs nerve conduits were similar to those of the autografts. The GGT nerve conduit offers a better scaffold for the incorporation of seeding undifferentiated ADSCs, and opens a new avenue to replace autologous nerve grafts for the rapid regeneration of damaged peripheral nerve tissues and an improved approach to patient care.

Local Anesthetic Schwann Cell Toxicity Is Time and Concentration Dependent

Peripheral nerve blocks with local anesthetics (LAs) are commonly performed to provide surgical anesthesia or postoperative analgesia. Nerve injury resulting in persistent numbness or weakness is a potentially serious complication. Local anesthetics have previously been shown to damage neuronal and Schwann cells via several mechanisms. We sought to test the hypothesis that LAs are toxic to Schwann cells and that the degree of toxicity is directly related to the concentration of LA and duration of exposure. Intraneural injection of LAs has been shown to produce nerve injury. We sought to test the hypothesis that a prolonged extraneural infusion of LA can also produce injury. Schwann cells cultured from neonatal rat sciatic nerves were incubated with LAs at different concentrations (10, 100, 500, and 1000 μM), and each concentration was assessed for toxicity after 4, 24, 48 and 72 hours of exposure. Local anesthetics tested were lidocaine, mepivacaine, chloroprocaine, ropivacaine, and bupivacaine. Cell death was assessed by lactate dehydrogenase release measured by optical density.In a separate experiment, a microcatheter was placed along the sciatic nerves of Sprague-Dawley rats. Rats were randomly assigned to receive either 0.9% saline (n = 8) or bupivacaine (0.5%, n = 4; 0.75%, n = 4) via the perineural catheters for 72 hours. The rats were then killed, and their nerves sectioned and stained for analysis. Sections were stained for myelin and with an antimacrophage (CD68) antibody. None of the LAs tested produced significant Schwann cell death at very low concentrations (10 μM, or 0.0003%) even after prolonged exposure. With prolonged exposure (48 or 72 hrs) to high concentrations (1000 μM, or 0.03%), all of the LAs tested produced significant Schwann cell death (increased lactate dehydrogenase release relative to control as measured by optical density, 0.384-0.974; all P values < 0.001). Only bupivacaine produced significant cell death (0.482, P < 0.001) after prolonged exposure to low concentrations (100 μM, or 0.003%). At intermediate concentrations (500 μM, or 0.015%), cell death was more widespread with bupivacaine (0.768, P < 0.001) and ropivacaine (0.675, P < 0.001) than the other agents (0.204-0.368; all P values < 0.001). Prolonged extraneural exposure of rat sciatic nerves to bupivacaine caused significant demyelination and infiltration of nerves with inflammatory cells. Local anesthetics induce Schwann cell death in a time- and concentration-dependent manner. Bupivacaine and ropivacaine have greater toxicity at intermediate concentrations, and prolonged exposure to bupivacaine produces significant toxicity even at low concentrations. Brief exposure to high concentrations of bupivacaine damages Schwann cells. Prolonged extraneural infusion of bupivacaine results in nerve injury.

New 3D hyaluronan-based scaffold for in vitro reconstruction of the rat sciatic nerve

Objective: For peripheral nerve regeneration, three-dimensional distribution and growth of cells within the porous scaffold are of clinical significance. The purpose of this study was to test in vitro a novel hyaluronic acid-based tubular conduit (HYAFF-11 biomaterials: 1 × 10 mm) as a nerve guide.Methods: Human fibroblasts, RN22 Schwann cell lines, human umbilical vein endothelial cells and primary nerve cells, obtained from neonatal rat sciatic nerve, were harvested and seeded on HYAFF-11 devices. Histologic (hematoxylin–eosin), immunohistochemical (antibodies to S100, CD31 and Von Willebrand factor) and PCR analyses were performed after 7 and 14 days from cell seeding onto biomaterials. MTT-based (thiazolyl blue) and DELFIA cell proliferation kit tests were performed to observe the biocompatibility of the cells cultured within the biomaterial devices.Results: We concluded that the conduits were not cytotoxic and demonstrated that cultured RN22 Schwann cells and rat Schwann cells grow in vitro on new artificial nerve conduits. We thus inferred that the HYAFF-11 conduit was a suitable biomaterial able to support nerve cell growth in vitro and after 14 days of cultivation, remained circular with a round lumen, maintaining the size and shape of its original architecture. Finally, attachment and proliferation of endothelial cells attested to the feasibility of developing a coculture system to promote in vivo integration of a microvascularized nerve substitute.Discussion: HYAFF-11 pre-seeded with Schwann and endothelial cells has the potential to be an alternative to autografting for the repair of long peripheral nerve defects.

FA2H is responsible for the formation of 2-hydroxy galactolipids in peripheral nervous system myelin

Myelin in the mammalian nervous system has a high concentration of galactolipids [galactosylceramide (GalCer) and sulfatide] with 2-hydroxy fatty acids. We recently reported that fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is the major fatty acid 2-hydroxylase in the mouse brain. In this report, we show that FA2H also plays a major role in the formation of 2-hydroxy galactolipids in the peripheral nervous system. FA2H mRNA and FA2H activity in the neonatal rat sciatic nerve increased rapidly during developmental myelination. The contents of 2-hydroxy fatty acids were approximately 5% of total galactolipid fatty acids at 4 days of age and increased to 60% in GalCer and to 35% in sulfatides at 60 days of age. The chain length of galactolipid fatty acids also increased significantly during myelination. FA2H expression in cultured rat Schwann cells was highly increased in response to dibutyryl cyclic AMP, which stimulates Schwann cell differentiation and upregulates myelin genes, such as UDP-galactose:ceramide galactosyltransferase and protein zero. These observations indicate that FA2H is a myelination-associated gene. FA2H-directed RNA interference (RNAi) by short-hairpin RNA expression resulted in a reduction of cellular 2-hydroxy fatty acids and 2-hydroxy GalCer in D6P2T Schwannoma cells, providing direct evidence that FA2H-dependent fatty acid 2-hydroxylation is required for the formation of 2-hydroxy galactolipids in peripheral nerve myelin. Interestingly, FA2H-directed RNAi enhanced the migration of D6P2T cells, suggesting that, in addition to their structural role in myelin, 2-hydroxy lipids may greatly influence the migratory properties of Schwann cells.

Acetylcholine inhibits cell cycle progression in rat Schwann cells by activation of the M2 receptor subtype

Cultures of Schwann cells from neonatal rat sciatic nerves were treated with acetylcholine agonists and the effects on cell proliferation evaluated. (3)[H]-thymidine incorporation shows that acetylcholine (ACh) receptor agonists inhibit cell proliferation, and FACS analysis demonstrates cell-cycle arrest and accumulation of cells in the G1 phase. The use of arecaidine, a selective agonist of muscarinic M2 receptors reveals that this effect depends mainly on M2 receptor activation. The arecaidine dependent-block in G1 is reversible because removal of arecaidine from the culture medium induces progression to the S phase. The block of the G1-S transition is also characterized by modulation of the expression of several cell-cycle markers. Moreover, treatment with ACh receptor agonist causes both a decrease in the PCNA protein levels in Schwann cell nuclei and an increase in p27 and p53 proteins. Finally, immuno-electron microscopy demonstrates that M2 receptors are expressed by Schwann cells in vivo. These results indicate that ACh, by modulating Schwann cell proliferation through M2 receptor activation, might contribute to their progression to a more differentiated phenotype.

ATF3 upregulation in glia during Wallerian degeneration: differential expression in peripheral nerves and CNS white matter

BackgroundMany changes in gene expression occur in distal stumps of injured nerves but the transcriptional control of these events is poorly understood. We have examined the expression of the transcription factors ATF3 and c-Jun by non-neuronal cells during Wallerian degeneration following injury to sciatic nerves, dorsal roots and optic nerves of rats and mice, using immunohistochemistry and in situ hybridization.ResultsFollowing sciatic nerve injury – transection or transection and reanastomosis – ATF3 was strongly upregulated by endoneurial, but not perineurial cells, of the distal stumps of the nerves by 1 day post operation (dpo) and remained strongly expressed in the endoneurium at 30 dpo when axonal regeneration was prevented. Most ATF3+ cells were immunoreactive for the Schwann cell marker, S100. When the nerve was transected and reanastomosed, allowing regeneration of axons, most ATF3 expression had been downregulated by 30 dpo. ATF3 expression was weaker in the proximal stumps of the injured nerves than in the distal stumps and present in fewer cells at all times after injury. ATF3 was upregulated by endoneurial cells in the distal stumps of injured neonatal rat sciatic nerves, but more weakly than in adult animals. ATF3 expression in transected sciatic nerves of mice was similar to that in rats. Following dorsal root injury in adult rats, ATF3 was upregulated in the part of the root between the lesion and the spinal cord (containing Schwann cells), beginning at 1 dpo, but not in the dorsal root entry zone or in the degenerating dorsal column of the spinal cord. Following optic nerve crush in adult rats, ATF3 was found in some cells at the injury site and small numbers of cells within the optic nerve displayed weak immunoreactivity. The pattern of expression of c-Jun in all types of nerve injury was similar to that of ATF3.ConclusionThese findings raise the possibility that ATF3/c-Jun heterodimers may play a role in regulating changes in gene expression necessary for preparing the distal segments of injured peripheral nerves for axonal regeneration. The absence of the ATF3 and c-Jun from CNS glia during Wallerian degeneration may limit their ability to support regeneration.

A quantitative morphometric analysis of rat spinal cord remyelination following transplantation of allogenic Schwann cells.

Quantitative morphometric techniques were used to assess the extent and pattern of remyelination produced by transplanting allogenic Schwann cells into demyelinated lesions in adult rat spinal cords. The effects of donor age, prior culturing of donor cells, prior lesioning of donor nerves, and host immunosuppression were evaluated by transplanting suspensions of 30,000 acutely dissociated or cultured Schwann cells from neonatal, young adult, or aged adult rat sciatic nerves into X-irradiation and ethidium bromide-induced demyelinated dorsal column lesions, with or without co-transplantation of neonatal optic nerve astrocytes. Three weeks after transplantation, spinal cords were processed for histological analysis. Under all Schwann cell transplant protocols, large areas containing many Schwann cell-like myelinated axon profiles could be readily observed throughout most of the lesion length. Within these "myelin-rich" regions, the vast majority of detectable axons showed a peripheral-like pattern of myelination. However, interaxonal spacing also increased, resulting in densities of myelinated axons that were more similar to peripheral nerve than intact dorsal columns. Freshly isolated Schwann cells remyelinated more axonal length than cultured Schwann cells, and cells from younger donors remyelinated slightly more axon length than cells from older donors, but all Schwann cell transplant protocols remyelinated tens of thousands of millimeters of axon length and remyelinated axons at similar densities. These results indicate that Schwann cells prepared under a variety of conditions are capable of eliciting remyelination, but that the density of remyelinated axons is much lower than the myelinated axon density in intact spinal cords.

Rapid effects of triiodothyronine on immediate-early gene expression in Schwann cells.

In the peripheral nervous system, triiodothyronine (T3) plays an important role in the development and regeneration of nerve fibers and in myelin formation. However, the target genes of T3 in peripheral nerves remain to be identified. We investigated whether T3 activated genes of transcription factors in Schwann cells. Expression of egr-1 (krox-24), egr-2 (krox-20), egr-3, c-jun, junB, c-fos, fosB, fra-1, fra-2, and CREB genes was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in Schwann cells isolated from neonatal rat sciatic nerves and in the cell lines MSC-80 (mouse Schwann cells), NIH-3T3 (mouse fibroblasts), and CHO (Chinese hamster ovary cells). Some of these transcription factors have been shown to be involved in Schwann cell differentiation. T3 triggered a rapid (15-30 min), transient (1-2-h) and strong (6- to 15-fold) stimulation of Egr-1, Egr-2, Egr-3, Jun B, c-Fos, and Fos B mRNA expression in Schwann cells. In contrast, expression of c-Jun, Fra-1, Fra-2, and CREB mRNA was not affected by T3. The stimulatory effects of T3 could be abolished by adding actinomycin D. T3 triggered the same pattern of gene stimulation in the mouse Schwann cell line MSC80, but not in the NIH-3T3 and CHO cell lines. Serum activated all the genes that responded to T3 and in addition fra-1 and fra-2, but not c-jun and CREB. Immunoblotting showed that the increase in Egr-1 and c-Fos mRNA levels was accompanied by an increase in the corresponding proteins. In addition, shifts of the protein bands indicated a posttranslational modification of the two proteins. These effects of T3 are likely to be mediated by the intracellular T3 receptor, as the D-isomer RT3 and T0, which do not bind to T3 receptors, proved ineffective. The present data suggested that T3 may regulate Schwann cell functions and differentiation by transiently activating the expression of specific transcription factors.

Progesterone stimulates Krox-20 gene expression in Schwann cells

The gene of the zinc finger transcription factor Krox-20 (Egr-2) is expressed in Schwann cells and plays an important role in myelination of peripheral nerves. We have shown that progesterone promotes myelination in the regenerating sciatic nerve and in cocultures of Schwann cells and sensory neurones. To determine whether progesterone regulates Krox-20 expression, we measured its effects on Krox-20 mRNA levels in the MSC80 mouse Schwann cell line by semi-quantitative RT-PCR. Although low levels of Krox-20 mRNA are detectable in MSC80 cells cultured in defined medium, treatment with 10 −6 M progesterone induces a rapid (15 min) and transient increase in the levels of Krox-20 mRNA. Lower doses of progesterone (10 −9, 10 −8 and 10 −7 M) are also effective in increasing Krox-20 mRNA. Other steroids including testosterone, dexamethasone, and estradiol are ineffective when added to the culture medium at 10 −6 M for 1 h. The induction of Krox-20 mRNA was also observed with the selective progesterone agonist Organon 2058 and was abolished by treating the MSC80 Schwann cells with the progesterone antagonist RU486, indicating that progesterone induces Krox-20 mRNA expression by binding to its intracellular receptor. The induction of Krox-20 by progesterone was also demonstrated in primary cultures of Schwann cells isolated from neonatal rat sciatic nerves, at the mRNA level by RT-PCR and at the protein level by immunohistochemistry. As Krox-20 is a necessary step for the initiation of myelin formation in peripheral nerves, its stimulation by progesterone suggests an important signalling function for this steroid in myelination.

Synthesis of progesterone in Schwann cells: regulation by sensory neurons.

In peripheral nerves, progesterone synthesized by Schwann cells has been implicated in myelination. In spite of such an important function, little is known of the regulation of progesterone biosynthesis in the nervous system. We show here that in rat Schwann cells, expression of the 3 beta-hydroxysteroid dehydrogenase and formation of progesterone are dependent on neuronal signal. Levels of 3 beta-hydroxysteroid dehydrogenase mRNA and synthesis of [3H]progesterone from [3H]pregnenolone were low in purified Schwann cells prepared from neonatal rat sciatic nerves. However, when Schwann cells were cultured in contact with sensory neurons, both expression and activity of the 3 beta-hydroxysteroid dehydrogenase were induced. Regulation of 3 beta-hydroxysteroid dehydrogenase expression by neurons was also demonstrated in vivo in the rat sciatic nerve. 3 beta-hydroxysteroid dehydrogenase mRNA was present in the intact nerve, but could no longer be detected 3 or 6 days after cryolesion, when axons had degenerated. After 15 days, when Schwann cells made new contact with the regenerating axons, the enzyme was re-expressed. After nerve transection, which does not allow axonal regeneration, 3 beta-hydroxysteroid dehydrogenase mRNA remained undetectable. The regulation of 3 beta-hydroxysteroid dehydrogenase mRNA after lesion was similar to the regulation of myelin protein zero (P0) and peripheral myelin protein 22 (PMP22) mRNAs, supporting an important role of locally formed progesterone in myelination.

Schwann Cells Transplanted into Normal and X-Irradiated Adult White Matter Do Not Migrate Extensively and Show Poor Long-Term Survival

Although Schwann cells are able to enter the central nervous system (CNS) when the integrity of the glia limitans is disrupted, their ability to migrate through intact CNS remains unclear. We have addressed this issue by transplanting lacZ-labeled Schwann cells into normal adult spinal cord white matter, and into X-irradiated spinal cord (an environment that, unlike normal spinal cord, permits the migration of transplanted oligodendrocyte progenitors). Schwann cell cultures, obtained from neonatal rat sciatic nerve and expanded using bovine pituitary extract and forskolin, were transfected by repeated exposure to retroviral vectors encoding the Escherichia coli lacZ gene. The normal behavior of the transduced cells was confirmed by transplantation into a nonrepairing area of demyelination in the spinal cord, where they formed myelin sheaths around demyelinated axons. A single microliter containing 4 × 104 cells was then transplanted into unlesioned normal and X-irradiated white matter of the spinal cord of adult syngeneic rats. One hour after injection, blue cells were observed as a discrete mass within the dorsal funiculus with a longitudinal distribution of 2–3 mm, indicating the extent of passive spread of the injected cells. At subsequent survival times (1, 2, and 4 weeks posttransplantation) blue cells had a distribution that was no more extensive than that seen 1 h after transplantation. However, the number of Schwann cells declined with time following transplantation such that at 4 weeks there were few surviving Schwann cells in both X-irradiated and nonirradiated spinal cord. These results indicate that transplanted Schwann cells do not migrate extensively and show poor long-term survival when introduced into a normal CNS environment.

Schwann cell remyelination is restricted to astrocyte-deficient areas after transplantation into demyelinated adult rat brain.

The ability to generate large numbers of Schwann cells from a peripheral nerve biopsy makes them potential candidates for the clinical application of cell transplantation to enhance remyelination in human demyelinating disease. Transplant-derived Schwann cell remyelination has previously been demonstrated in the spinal cord but not for demyelinated axons in the brain, a more likely site for initial clinical intervention. We have transplanted Schwann cells from male neonatal rat sciatic nerves into ethidium bromide-induced areas of demyelination in the deep cerebellar white matter of adult female rats. The extent of Schwann cell remyelination 28 days after transplantation was significantly increased in lesions that received direct injections of Schwann cells compared with non-transplanted lesions. Using in situ hybridisation to identify the rat Y chromosome, transplanted male cells were found to co-localise with the P0 immunoreactive area of Schwann cell remyelination. Combined immunohistochemistry and in situ hybridisation confirmed that many remyelinating Schwann cells were transplant-derived. P0 immunoreactivity and transplanted male cells were found in GFAP-negative, astrocyte-free areas. Transplanted Schwann cells were not identified outside of transplanted lesions, nor did they did not contribute to remyelination of a lesion at a distance from the site of transplantation. Our findings indicate that demyelinated axons in the adult brain can be remyelinated by transplanted Schwann cells but that migration and remyelination are restricted to areas from which astrocytes are absent.

Areas of demyelination do not attract significant numbers of schwann cells transplanted into normal white matter.

If Schwann cell transplantation is to be used as a therapy for demyelinating disease, it is important to know if the number of transplanted cells and their transplantation site affects the extent of remyelination. Primary Schwann cell cultures were obtained from neonatal rat sciatic nerve, purified, and expanded using bovine pituitary extract and forskolin. Areas of persistent demyelination were created in the dorsal funiculus of the thoracolumbar spinal cord of rats by injecting ethidium bromide into white matter exposed to 40 Gy of X-irradiation, and a high and low number of Schwann cells were transplanted, into either the area of demyelination or the dorsal funiculus cranial to the area of demyelination. Animals were perfused 4 weeks after transplantation. After injection of 4 x 10(4) cells into the area of demyelination, the area of Schwann cell remyelination was 0.88 +/- 0.16 mm(2), while following the injection of 3 x 10(3) cells it was significantly smaller, 0.29 +/- 0.09 mm(2). After implantation of Schwann cells 1-3 mm (mean 2.5 mm) cranial to the area of demyelination, only one of the eight animals (a high-dose animal) showed extensive Schwann cell remyelination. In this animal, the cells were transplanted within 1 mm of the area of demyelination, well within the length of tissue over which cells are passively spread by the injection procedure (1-3 mm). Our results show that significant numbers of transplanted Schwann cells are not attracted through normal tissue to areas of demyelination and when transplanted into areas of demyelination the extent of myelination is related to the number of Schwann cells transplanted.

Sensory and Motor Neuron-derived Factor Is a Transmembrane Heregulin That Is Expressed on the Plasma Membrane with the Active Domain Exposed to the Extracellular Environment

The beta-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the beta-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other beta-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl-terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor.

Integration of Transplanted Cultured Schwann Cells into the Long Myelinated Fiber Tracts of the Adult Spinal Cord

A suspension of about 10,000 purified Schwann cells cultured from the neonatal rat sciatic nerve was transplanted into a discrete site in the upper cervical level of the corticospinal tract of one side in adult rats. From 4 days after transplantation immunostaining for p75 (low-affinity neurotrophin receptor) showed that the transplants consisted of a central mass of Schwann cells and cuffs of elongated Schwann cells along the perivascular space of curving blood vessels (most of which had been formed in response to the transplantation). Schwann cells leaving the central mass and perivascular cuffs migrated in strictly linear orientation along the rostrocaudal axis of the host corticospinal tract. According to the territory through which they migrated, the transplanted Schwann cells adopted two quite different forms: (1) Therow Schwann cells,which migrated singly or in groups within the rows of host oligodendrocytic and astrocytic cell bodies, were non-process-bearing, rather cuboidal, brick-like cells (about 8×12 μm in size). (2) In contrast, theinterfascicular Schwann cells,which migrated singly or intertwined in rope-like small groups interspersed among the axons of the host corticospinal tract, were larger, symmetrically bipolar cells, with processes about 100–120 μm long and 2 μm wide and bulging, ovoid nuclei, located in centrally placed cell bodies about 10 μm across. After about 6 weeks, the p75 immunoreactivity of the interfascicular Schwann cells had become downregulated. However, from as early as 10 days after transplantation, immunostaining for the peripheral myelin protein, P0, semithin sections, and electron microscopy showed that these Schwann cells were not lost, but that they had myelinated the segments of the host corticospinal axons in the region of the transplant. In contrast, the row Schwann cells did not express P0or form myelin. They retained their p75 immunoreactivity at long survivals (presumably because they were secluded from contacting the tract axons). The row Schwann cells also migrated farther than the interfascicular Schwann cells (possibly a function of their maintained p75 expression), becoming dispersed singly for at least 8 mm from the original transplant site. Our previous study of corticospinal tract lesions had shown the formation of a “closed” scar formed by hypertrophic astrocytic processes, which walled off a central astrocyte-free region and totally disrupted the normal longitudinal alignment of the tract astrocytic processes. In contrast, while the present Schwann cell transplants induced a comparable astrocytic hypertrophy over the same time course, the astrocytic processes remained able to penetrate the transplant site, which was not walled off, so that the longitudinal arrangement of the host corticospinal tract astrocytic skeleton was preserved intact across the region of the transplant. These observations show that Schwann cells can be intimately integrated into the cytoarchitecture of the myelinated adult host corticospinal tract. This integration is not a random dispersal in damaged areas: it involves direct interaction with the cell elements present in the host tract, it respects the complex and regular organization of the host tract glial cells, and it results in the formation of a precisely arranged mosaic of central and peripheral tissue.