Mouse Peripheral Nerve Research Articles

Sulfatide decrease in myelin influences formation of the paranodal axo‐glial junction and conduction velocity in the sciatic nerve

Cerebroside sulfotransferase (CST) catalyzes the production of sulfatide, which is one of the major glycolipids in myelin. Homozygous CST knockout mice were shown to be completely deficient in sulfatide. They were born healthy but began to display progressive neurological deficits from 6 weeks of age. Severe abnormalities of paranodal regions and changes in axonal ion channel distribution were prominent in both the central and peripheral nervous systems. But whether partial decreases in myelin sulfatide levels influence paranodal formation, as well as nerve conduction velocity (NCV), is largely unknown. To determine the functional significance of sulfatide content in myelin, we performed electrophysiological, morphological, and biochemical analyses using heterozygote, homozygote, and wild-type mouse peripheral nerves and compared the results with individual sulfatide content. NCVs were significantly reduced in homozygote animals compared with wild-type mice. In contrast, these values were markedly varied in individual heterozygote mice. On the basis of NCV values, we divided heterozygous mice into two groups: mice with mild but significant reduction of NCV and those with normal NCV. Teased nerve fibers obtained from individual mouse sciatic nerves were immunostained, and Na(+) channel and Caspr cluster lengths were measured to determine abnormal levels of junctional formation at the paranode. Furthermore, sulfatide content in each sciatic nerve was examined by thin layer chromatography. The results demonstrated significant correlations among sulfatide level, severity of paranodal abnormality, and reduction of NCV. Thus, the fine regulation of myelin sulfatide content by CST is important for normal function of myelinated axons.

Distribution of Podoplanin-Expressing Cells in the Mouse Nervous Systems

Podoplanin is a mucin-type glycoprotein which was first identified in podocytes. Recently, podoplanin has been successively reported as a marker for brain and peripheral nerve tumors, however, the distribution of podoplanin-expressing cells in normal nerves has not been fully investigated. This study aims to examine the podoplanin-expressing cell distribution in the mouse head and nervous systems. An immunohistochemical study showed that the podoplanin-positive areas in the mouse peripheral nerve and spinal cord are perineurial fibroblasts, satellite cells in the dorsal root ganglion, glia cells in the ventral and dorsal horns, and schwann cells in the ventral and dorsal roots; in the cranial meninges the dura mater, arachnoid, and pia mater; in the eye the optic nerve, retinal pigment epithelium, chorioidea, sclera, iris, lens epithelium, corneal epithelium, and conjunctival epithelium. In the mouse brain choroid plexus and ependyma were podoplanin-positive, and there were podoplanin-expressing brain parenchymal cells in the nuclei and cortex. The podoplanin-expressing cells were astrocyte marker GFAP-positive and there were no differences in the double positive cell distribution of several portions in the brain parenchyma except for the fornix. The results suggest that podoplanin may play a common role in nervous system support cells and eye constituents.

Distribution of serine/threonine kinase SAD-B in mouse peripheral nerve synapse

The serine/threonine kinase SAD regulates neural functions such as axon/dendrite polarization and neurotransmitter release. In the vertebrate central nervous system, SAD-B, a homolog of Caenorhabditis elegans SAD-1, is associated with synaptic vesicles and the active zone cytomatrix in nerve terminals. However, the distribution of SAD-B in the peripheral nervous system remains elusive. Here, we show that SAD-B is specifically localized to neuromuscular junctions. Although the active zone protein bassoon showed a punctated signal indicating its localization to motor end plates, SAD-B shows relatively diffuse localization indicating its association with both the active zone and synaptic vesicles. Therefore, SAD kinase may regulate neurotransmitter release from motor end plates in a similar manner to its regulation of neurotransmitter release in the central nervous system.

181 CELL THERAPY WITH AUTOLOGOUS URINE-DERIVED STEM CELLS FOR VESICOURETERAL REFLUX

You have accessJournal of UrologyStem Cell Research1 Apr 2011181 CELL THERAPY WITH AUTOLOGOUS URINE-DERIVED STEM CELLS FOR VESICOURETERAL REFLUX Shaofeng Wu, GuiHua Liu, Shantaram Bharadwaj, Steve Hoagie, Anthony Atala, and Yuanyuan Zhang Shaofeng WuShaofeng Wu Winston-Salem, NC More articles by this author , GuiHua LiuGuiHua Liu Winston-Salem, NC More articles by this author , Shantaram BharadwajShantaram Bharadwaj Winston-Salem, NC More articles by this author , Steve HoagieSteve Hoagie Winston-Salem, NC More articles by this author , Anthony AtalaAnthony Atala Winston-Salem, NC More articles by this author , and Yuanyuan ZhangYuanyuan Zhang Winston-Salem, NC More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.250AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES As an alternative to bulking agents, endoscopic injections of autologous cells have been used to restore the normal structure and function of the vesicoureteral junction. Vascularization of the injected graft is critical for enhancement of cell survival and maintenance of the volume of the graft. Recently, we have demonstrated that certain cells in urine possess the characteristics of stem cells, including extensive expansion capability and multipotency. The goal of this study was to evaluate the effect of vascular endothelial growth factor (VEGF) over-expression on urine-derived stem cell (USC) survival, growth and myogenic differentiation and to determine whether this technique could improve injection therapy for the correction of VUR. METHODS USC were obtained from 9 urine samples (5 healthy individual donors; ages 3 to 27). USC were infected with adenovirus containing the human VEGF gene (USC/Ad-VEGF). USC/Ad-VEGF plus endothelial cells (in total 5×106 cells) in 500μl collagen-I gel were subcutaneously implanted into 20 athymic mice. After 28 days, the grafts were assessed grossly and with histology and immunocytochemistry for smooth muscle markers (α-smooth muscle actin, desmin and myosin) endothelial markers (CD 31 and von Willeband factor) and peripheral nerve cell markers (S-100, neurofilament, glial fibrillary acidic protein). RESULTS The grafts containing both USC/Ad-VEGF and endothelial cells were larger and better vascularized compared to non-VEGF expressing controls. Additionally, the total number of implanted cells expressing human nuclear markers was significantly higher, indicating increased cell survival in the VEGF-expressing grafts. More VEGF-expressing cells expressed CD31 and von Willebrand proteins, as well as smooth muscle markers. Interestingly, more nerve fibers and revascularization appeared in the USC/Ad-VEGF plus endothelial cells group than the controls in vivo. However, the regenerated nerve cells did not stain for human nuclear protein expression, indicating that these regenerated nerve fibers most likely originated from mouse peripheral nerve rather than from the differentiation of USC. CONCLUSIONS VEGF over-expression in grafted USC enhanced the in vivo survival of these cells and increased neovascularization, myogenic differentiation of USC, and nerve regeneration within the grafts. This approach might have important clinical implications for urological cell therapy for the treatment of VUR and urinary incontinence. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e75 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shaofeng Wu Winston-Salem, NC More articles by this author GuiHua Liu Winston-Salem, NC More articles by this author Shantaram Bharadwaj Winston-Salem, NC More articles by this author Steve Hoagie Winston-Salem, NC More articles by this author Anthony Atala Winston-Salem, NC More articles by this author Yuanyuan Zhang Winston-Salem, NC More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Diffusion tensor imaging to assess axonal regeneration in peripheral nerves

Development of outcome measures to assess ongoing nerve regeneration in the living animal that can be translated to human can provide extremely useful tools for monitoring the effects of therapeutic interventions to promote nerve regeneration. Diffusion tensor imaging (DTI), a magnetic resonance based technique, provides image contrast for nerve tracts and can be applied serially on the same subject with potential to monitor nerve fiber content. In this study, we examined the use of ex vivo high-resolution DTI for imaging intact and regenerating peripheral nerves in mice and correlated the MRI findings with electrophysiology and histology. DTI was done on sciatic nerves with crush, without crush, and after complete transection in different mouse strains. DTI measures, including fractional anisotropy (FA), parallel diffusivity, and perpendicular diffusivity were acquired and compared in segments of uninjured and crushed/transected nerves and correlated with morphometry. A comparison of axon regeneration after sciatic nerve crush showed a comparable pattern of regeneration in different mice strains. FA values were significantly lower in completely denervated nerve segments compared to uninjured sciatic nerve and this signal was restored toward normal in regenerating nerve segments (crushed nerves). Histology data indicate that the FA values and the parallel diffusivity showed a positive correlation with the total number of regenerating axons. These studies suggest that DTI is a sensitive measure of axon regeneration in mouse models and provide basis for further development of imaging technology for application to living animals and humans.

Treadmill training enhances axon regeneration in injured mouse peripheral nerves without increased loss of topographic specificity.

We investigated the extent of misdirection of regenerating axons when that regeneration was enhanced by using treadmill training. Retrograde fluorescent tracers were applied to the cut proximal stumps of the tibial and common fibular nerves 2 or 4 weeks after transection and surgical repair of the mouse sciatic nerve. The spatial locations of retrogradely labeled motoneurons were studied in untreated control mice and in mice receiving 2 weeks of treadmill training, according to either a continuous protocol (10 m/minute, 1 hour/day, 5 days/week) or an interval protocol (20 m/minute for 2 minutes, followed by a 5-minute rest, repeated four times, 5 days/week). More retrogradely labeled motoneurons were found in both treadmill-trained groups. The magnitude of this increase was as great as or greater than that found after using other enhancement strategies. In both treadmill-trained groups, the proportions of motoneurons labeled from tracer applied to the common fibular nerve that were found in spinal cord locations reserved for tibial motoneurons in intact mice were no greater than in untreated control mice and significantly less than those found after electrical stimulation or chondroitinase treatment. Treadmill training in the first 2 weeks following peripheral nerve injury produces a marked enhancement of motor axon regeneration without increasing the propensity of those axons to choose pathways leading to functionally inappropriate targets.

Immune Responses Following Mouse Peripheral Nerve Xenotransplantation in Rats

Xenotransplantation offers a potentially unlimited source for tissues and organs for transplantation, but the strong xenoimmune responses pose a major obstacle to its application in the clinic. In this study, we investigate the rejection of mouse peripheral nerve xenografts in rats. Severe intragraft mononuclear cell infiltration, graft distension, and necrosis were detected in the recipients as early as 2 weeks after mouse nerve xenotransplantation. The number of axons in xenografts reduced progressively and became almost undetectable at week 8. However, mouse nerve xenotransplantation only led to a transient and moderate increase in the production of Th1 cytokines, including IL-2, IFN-γ, and TNF-α. The data implicate that cellular immune responses play a critical role in nerve xenograft rejection but that further identification of the major effector cells mediating the rejection is required for developing effective means to prevent peripheral nerve xenograft rejection.

Deletion of p75NTR impairs regeneration of peripheral nerves in mice

Aims After peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves. Main methods In p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry. Key findings The results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice. Significance Our data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.

Tumor Suppressor Schwannomin/Merlin Is Critical for the Organization of Schwann Cell Contacts in Peripheral Nerves

Schwannomin/merlin is the product of a tumor suppressor gene mutated in neurofibromatosis type 2 (NF2). Although the consequences of NF2 mutations on Schwann cell proliferation are well established, the physiological role of schwannomin in differentiated cells is not known. To unravel this role, we studied peripheral nerves in mice overexpressing in Schwann cells schwannomin with a deletion occurring in NF2 patients (P0-SCH-Delta39-121) or a C-terminal deletion. The myelin sheath and nodes of Ranvier were essentially preserved in both lines. In contrast, the ultrastructural and molecular organization of contacts between Schwann cells and axons in paranodal and juxtaparanodal regions were altered, with irregular juxtaposition of normal and abnormal areas of contact. Similar but more severe alterations were observed in mice with conditional deletion of the Nf2 gene in Schwann cells. The number of Schmidt-Lanterman incisures, which are cytoplasmic channels interrupting the compact myelin and characterized by distinct autotypic contacts, was increased in the three mutant lines. P0-SCH-Delta39-121 and conditionally deleted mice displayed exuberant wrapping of nonmyelinated fibers and short internodes, an abnormality possibly related to altered control of Schwann cell proliferation. In support of this hypothesis, Schwann cell number was increased along fibers before myelination in P0-SCH-Delta39-121 mice but not in those with C-terminal deletion. Schwann cell numbers were also more numerous in mice with conditional deletion. Thus, schwannomin plays an important role in the control of Schwann cell number and is necessary for the correct organization and regulation of axoglial heterotypic and glio-glial autotypic contacts.

Presynaptic and Postsynaptic Amplifications of Neuropathic Pain in the Anterior Cingulate Cortex

Neuropathic pain is caused by a primary lesion or dysfunction in the nervous system. Investigations have mainly focused on the spinal mechanisms of neuropathic pain, and less is known about cortical changes in neuropathic pain. Here, we report that peripheral nerve injury triggered long-term changes in excitatory synaptic transmission in layer II/III neurons within the anterior cingulate cortex (ACC). Both the presynaptic release probability of glutamate and postsynaptic glutamate AMPA receptor-mediated responses were enhanced after injury using the mouse peripheral nerve injury model. Western blot showed upregulated phosphorylation of GluR1 in the ACC after nerve injury. Finally, we found that both presynaptic and postsynaptic changes after nerve injury were absent in genetic mice lacking calcium-stimulated adenylyl cyclase 1 (AC1). Our studies therefore provide direct integrative evidence for both long-term presynaptic and postsynaptic changes in cortical synapses after nerve injury, and that AC1 is critical for such long-term changes. AC1 thus may serve as a potential therapeutic target for treating neuropathic pain.

Comparison of blood–nerve barrier disruption and matrix metalloprotease-9 expression in injured central and peripheral nerves in mice

To investigate the involvement of blood-born factors and extracellular proteases in axonal degeneration and regeneration in both PNS and CNS, we directly compared the differences of blood-nerve barrier (BNB) disruption and matrix metalloprotease-9 (MMP-9) induction between the sciatic nerve and optic nerve after crush injury in the same animal. In sciatic nerve, BNB disruption, fibrin(ogen) deposition and MMP-9 expression were observed only in the first week following injury. Neurofilament (NF) immunoreactivity dramatically decreased in the first 2 days, gradually recovered to the normal levels by day 28. In contrast, the immunoglobulin G deposits spanned from 4 h to 28 days in crushed optic nerves. Fibrin(ogen) deposition was only observed in the first 2 days, while MMP-9 induction did not occur until a week after injury but lasted for 3 weeks in the crushed optic nerves. The NF immunoreactivity did not change much until day 7 and almost completely disappeared on day 28. The decrease of NF immunoreactivity coincided with the induction of MMP-9 after optic nerve crush. These results show that BNB disruption and MMP-9 induction are differentially regulated in the PNS and CNS after injuries, and they may contribute to the different regeneration capacities of the two systems.

Histochemical demonstration of a monocarboxylate transporter in the mouse perineurium with special reference to GLUT1

Peripheral nerves express GLUT1 in both endoneurial blood vessels and the perineurium and utilize glucose as a major energy substrate, as does the brain. However, under conditions of a reduced utilization of glucose, the brain is dependent upon monocarboxylates such as ketone bodies and lactate, being accompanied by an elevated expression of a monocarboxylate transporter (MCT1) in the blood-brain barrier. The present immunohistochemical study aimed to examine the expression of MCT1 in the peripheral nerves of mice. MCT1 immunoreactivity was found in the perineurial sheath and colocalized with GLUT1, while the endoneurial blood vessels expressed GLUT1 only. An intense expression of MCT1 in the perineurium was confirmed by Western blot and in situ hybridization analyses. Ultrastructurally, the MCT1 and GLUT1 immunoreactivities in the thick perineurium showed an intensity gradient decreasing towards the innermost layer. In neonates, the MCT1 immunoreactivity in the perineurium was intense, while the GLUT1 immunoreactivity was faint or absent. These findings suggest that peripheral nerves depend on monocarboxylates as a major energy source and that MCT1 in the perineurium is responsible for the supply of monocarboxylates to nerve fibers and Schwann cells.

Mast cells can contribute to axon–glial dissociation and fibrosis in peripheral nerve

Expression of the human epidermal growth factor receptor (EGFR) in murine Schwann cells results in loss of axon-Schwann cell interactions and collagen deposition, modeling peripheral nerve response to injury and tumorigenesis. Mast cells infiltrate nerves in all three situations. We show that mast cells are present in normal mouse peripheral nerve beginning at 4 weeks of age, and that the number of mast-cells in EGFR(+) nerves increases abruptly at 5-6 weeks of age as axons and Schwann cells dissociate. The increase in mast cell number is preceded and accompanied by elevated levels of mRNAs encoding the mast-cell chemoattractants Rantes, SCF and VEGF. Genetic ablation of mast cells and bone marrow reconstitution in W(41) x EGFR(+) mice indicate a role for mast cells in loss of axon-Schwann cell interactions and collagen deposition. Pharmacological stabilization of mast cells by disodium cromoglycate administration to EGFR(+) mice also diminished loss of axon-Schwann cell interaction. Together these three lines of evidence support the hypothesis that mast cells can contribute to alterations in peripheral nerves.

FK506-Binding Protein Ligands: Structure-Based Design, Synthesis, and Neurotrophic/Neuroprotective Properties of Substituted 5,5-Dimethyl-2-(4-thiazolidine)carboxylates

Structure-based design and discovery of novel neuroimmunophilin FK506-binding protein (FKBP) ligands were pursued in the present study. The binding mode of the known FKBP ligand 1 (3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate) in complex with FKBP12 was investigated using LUDI simulation and upon which a novel scaffold structure predicted to possess improved binding affinity was designed. A virtual combinatorial library composed of diverse combinations of two substituted groups was constructed using Project Library, followed by an automated screening of the library against the ligand binding site on FKBP52 using DOCK. Forty-three candidate compounds that displayed favorable binding with the receptor were identified and synthesized. The neurotrophic activity of the candidate compounds was evaluated on chick dorsal root ganglion cultures in vitro. As a result, 15 compounds exhibited positive effects on ganglion neurite outgrowth in the presence of 0.15 ng/mL NGF, among which 7 compounds at testing concentrations of 1 pM and 100 pM showed greater efficacy than 1 at 100 pM. Compound 18 (3-(3-pyridyl)-1-propyl (2S)-5,5-dimethyl-1-(3,3-dimethyl-1,2-dioxobutyl)-2-(4-thiazolidine)carboxylate) afforded the most potent effect in promoting the processes of neurite outgrowth and which was in a concentration-dependent manner from 1 pM to 100 pM. Half-maximal effect occurred at about 10 pM. Moreover, 18 at a dosage of 10 mg/kg was found to be significantly neuroprotective in a mouse peripheral sympathetic nerve injury model induced by 8 mg/kg 6-hydroxydopamine. This study further suggests the clinical potential of novel FKBP ligands as a new therapeutic approach in the treatment of neurodegenerative disorders, such as Parkinson's disease.

Evaluation of Na+/K+ pump function following repetitive activity in mouse peripheral nerve

After conduction of prolonged trains of impulses the increased Na+/K+ pump activity leads to hyperpolarization. The aim of this study was to develop a mouse model to investigate the Na+/K+ pump function in peripheral nerve by measuring the decrease in excitability during activity-dependent hyperpolarization. Acute electrophysiological investigations were carried out in seven adult mice. Nerve excitability was evaluated by tracking the change in threshold current after 5min of 100Hz stimulation of the tibial nerve at ankle. We developed a threshold tracking system that allowed us to follow several excitability measures simultaneously from the evoked plantar compound muscle action potential (CMAP) and sciatic compound nerve action potential (CNAP). Three minutes after repetitive supramaximal stimulation maximal CMAP and CNAP amplitudes recovered but the threshold was increased ∼40% for motor axons ∼34% for axons generating CNAP. The threshold recovered with a rate of 3.8%/minute that was similar for nerve and motor responses. By tracking the effect of polarizing currents we found evidence of activity dependent hyperpolarization, and our data suggest that the observed threshold change after repetitive stimulation of the mouse tibial nerve is an indicator of the Na+/K+ pump function in vivo. Evaluation of activity-dependent hyperpolarization may be an important indicator of axonal ability to cope with Na+ load.

Disrupted Schwann cell–axon interactions in peripheral nerves of mice with altered L1-integrin interactions

The cell adhesion molecule L1 is important for peripheral nerve development. Mice lacking the 6th Ig domain of L1 (L1-6D mice) lose L1 homophilic binding and RGD dependent L1-integrin binding [Itoh, K., Cheng, L., Kamei, Y., Fushiki, S., Kamiguchi, H., Gutwein, P., Stoeck, A., Arnold, B., Altevogt, P., Lemmon, V., 2004. Brain development in mice lacking L1-L1 homophilic adhesion. J. Cell Biol. 165, 145-154]. We examined the ultrastructure of sciatic nerves from L1-6D at postnatal day 7 and 8 weeks. Unmyelinated axons frequently detached at the edge of Schwann cells, and naked axons were observed. Myelin was thinner in L1-6D and abnormal, multiple axons wrapped in a single myelin sheath were routinely observed. Previous work has shown that L1 on axons interacts with a heterophilic binding partner on Schwann cells to facilitate normal peripheral nerve formation. Taken together, it is likely that L1 on axons binds integrins on Schwann cells, resulting in interactions between axons and Schwann cells that are essential for ensheathment and myelination.

Study of neurotrophic activity of thrombin on the model of regenerating mouse nerve

Experiments demonstrated a dose-dependent facilitating effect of thrombin and peptide thrombin receptor agonist PAR1 (TRAP6) on regeneration of mouse peripheral nerve after its crushing. The maximum neurotrophic effect was observed at low concentrations of thrombin (10 nM) and TRAP6 (10 microM).

Inhibitory effect of cyclosporin A on p-Glycoprotein function in peripheral nerves of mice treated with doxorubicin and vinblastine

ObjectiveTo investigate the inhibitory effect of cyclosporin A on p-glycoprotein function in peripheral nerves (VIIth, VIIIth and sciatic nerves).Material and MethodsMale mdr1a(−/−) and mdr1a(+/+) FVB mice were used. Doxorubicin (30 mg/kg) was administered intravenously with or without i.p. administration of cyclosporin A (200 mg/kg). Vinblastine (5 mg/kg) was also administered intravenously with or without i.p. administration of cyclosporin A (200 mg/kg).ResultsTissue concentrations of doxorubicin and vinblastine in peripheral nerves of the mdr1a(+/+) mice pretreated with 200 mg/kg cyclosporin A were significantly higher than those in the mdr1a(+/+) mice administered doxorubicin or vinblastine alone, suggesting that cyclosporin A inhibited the efflux pump function of p-glycoprotein in the peripheral nerves. In the mdr1a(−/−) mice, tissue concentrations of doxorubicin and vinblastine in peripheral nerves were also significantly higher than those in the mdr1a(+/+) mice administered doxorubicin or vinblastine alone. Based on these results, it is suggested that p-glycoprotein plays an important role in blood–nerve barrier function by preventing side-effects induced by neurotoxic drugs.ConclusionWhen doxorubicin and vinblastine are co-administered with cyclosporin A, the patient should be carefully monitored because peripheral nerve disorders may be induced.

Magnetic cell sorting for enriching Schwann cells from adult mouse peripheral nerves

We have devised a simple method to purify mitotically active Schwann cells (SC) from peripheral nerves of adult mice. Nerves were predegenerated in vitro for 7 days and after dissociation cells were plated on poly- l-lysine/laminin coated dishes in N2 serum-free culture medium supplemented with forskolin and heregulin-β1. Primary cultures were purified from contaminating fibroblasts by magnetic cell sorting (MACS) based on SC membrane specific expression of p75 NGFR and enriched to about 99% of SC after MACS from 34 to 91% before sorting. After sorting, purified adult mouse SC were propagated for three passages until confluent to a total surface of 160 cm 2 per mouse (two sciatic and two trigeminal nerves). In addition, we show that this method can be used to purify tumoral SC from mouse NF2-related schwannomas.

Definition of pre- and postsynaptic forms of the CT carbohydrate antigen at the neuromuscular junction: ubiquitous expression of the CT antigens and the CT GalNAc transferase in mouse tissues

At the rodent neuromuscular junction, the synaptic expression of the CT carbohydrate antigens is defined by the binding of two monoclonal antibodies, CT1 and CT2. CT1 preferentially stains the presynaptic membrane, while CT2 preferentially stains the postsynaptic apparatus. Here we show that the differential subsynaptic distribution of these antigens is due to a preference of CT1 for structures containing N-acetyl neuraminic acid (NeuAc) and a preference of CT2 for structures containing N-glycolyl neuraminic acid (NeuGc). This was found to be the case both in binding to cultured myotubes, where NeuAc/NeuGc levels were manipulated by feeding acetylated N-acetyl mannosamine precursors, and in binding to purified GM2 ganglioside containing either NeuAc or NeuGc. At human neuromuscular junctions, where the enzymatic machinery to make NeuGc is absent [Proc. Natl. Acac. Sci. USA 95 (1998) 11751], CT1 and GM2(NeuAc) antibodies stained, while CT2 did not. Thus, the N-glycolyl modification of sialic acid helps to define the differential distribution of the CT antigens at the rodent neuromuscular junction, and this difference is lost in humans. In addition, sulfatase and 9- O-acetylesterase treatment of cells or tissues increased the amount of CT1 and CT2 antibody binding, with sulfatase differentially unmasking CT antigen expression on particular glycoproteins. Despite its uniquely synaptic localization in skeletal muscle, the CT antigens and the CT GalNAc transferase are ubiquitously expressed in other mouse tissues, including brain, spinal cord, and peripheral nerve. One of the proteins that can be co-purified with a CT-reactive glycoprotein is α dystroglycan. These data better define the sub-synaptic structures of the CT carbohydrate antigens at the neuromuscular junction and demonstrate their ubiquitous presence in mouse tissues, including the brain.