Sciatic Nerve Of Adult Mice Research Articles

Variation in the vulnerability of mice expressing human superoxide dismutase 1 to prion-like seeding: a study of the influence of primary amino acid sequence

Misfolded forms of superoxide dismutase 1 (SOD1) with mutations associated with familial amyotrophic lateral sclerosis (fALS) exhibit prion characteristics, including the ability to act as seeds to accelerate motor neuron disease in mouse models. A key feature of infectious prion seeding is that the efficiency of transmission is governed by the primary sequence of prion protein (PrP). Isologous seeding, where the sequence of the PrP in the seed matches that of the host, is generally much more efficient than when there is a sequence mis-match. Here, we used paradigms in which mutant SOD1 seeding homogenates were injected intraspinally in newborn mice or into the sciatic nerve of adult mice, to assess the influence of SOD1 primary sequence on seeding efficiency. We observed a spectrum of seeding efficiencies depending upon both the SOD1 expressed by mice injected with seeds and the origin of the seed preparations. Mice expressing WT human SOD1 or the disease variant G37R were resistant to isologous seeding. Mice expressing G93A SOD1 were also largely resistant to isologous seeding, with limited success in one line of mice that express at low levels. By contrast, mice expressing human G85R-SOD1 were highly susceptible to isologous seeding but resistant to heterologous seeding by homogenates from paralyzed mice over-expressing mouse SOD1-G86R. In other seeding experiments with G85R SOD1:YFP mice, we observed that homogenates from paralyzed animals expressing the H46R or G37R variants of human SOD1 were less effective than seeds prepared from mice expressing the human G93A variant. These sequence mis-match effects were less pronounced when we used purified recombinant SOD1 that had been fibrilized in vitro as the seeding preparation. Collectively, our findings demonstrate diversity in the abilities of ALS variants of SOD1 to initiate or sustain prion-like propagation of misfolded conformations that produce motor neuron disease.

Functional expression of glycine receptors in DRG neurons of mice

Glycine receptor is one of the chloride-permeable ion channels composed of combinations of four α subunits and one β subunit. In adult spinal cord, the glycine receptor α1 subunit is crucial for the generation of inhibitory neurotransmission. The reduced glycinergic inhibition is regarded as one of the key spinal mechanisms underlying pathological pain symptoms. However, the expression and function of glycine receptors in the peripheral system are largely unknown as yet. Here we found that glycine receptor α1 subunit was prevalent in the dorsal root ganglia (DRG) neurons as well as in the sciatic nerves of adult mice. Intraganglionar or intraplantar injection of glycine receptor antagonist strychnine caused the hypersensitivity to mechanical, thermal and cold stimuli, suggesting the functional importance of peripheral glycine receptors in the control of nociceptive signal transmission. Our data showed that peripheral inflammation induced by formalin decreased the expression of glycine receptor α1 subunit on the plasma membrane of DRG neurons, which was attributed to the activation of protein kinase C signaling. Intraplantar application of glycine receptor agonist glycine or positive modulator divalent zinc ion alleviated the first-phase painful behaviors induced by formalin. These data suggested that peripheral glycine receptor might serve as an effective target for pain therapy.

NTE/PNPLA6 is expressed in mature Schwann cells and is required for glial ensheathment of Remak fibers.

Neuropathy target esterase (NTE) or patatin-like phospholipase domain containing 6 (PNPLA6) was first linked with a neuropathy occurring after organophosphate poisoning and was later also found to cause complex syndromes when mutated, which can include mental retardation, spastic paraplegia, ataxia, and blindness. NTE/PNPLA6 is widely expressed in neurons but experiments with its Drosophila orthologue Swiss-cheese (SWS) suggested that it may also have glial functions. Investigating whether NTE/PNPLA6 is expressed in glia, we found that NTE/PNPLA6 is expressed by Schwann cells in the sciatic nerve of adult mice with the most prominent expression in nonmyelinating Schwann cells. Within Schwann cells, NTE/PNPLA6 is enriched at the Schmidt-Lanterman incisures and around the nucleus. When analyzing postnatal expression patterns, we did not detect NTE/PNPLA6 in promyelinating Schwann cells, while weak expression was detectable at postnatal day 5 in Schwann cells and increased with their maturation. Interestingly, NTE/PNPLA6 levels were upregulated after nerve crush and localized to ovoids forming along the nerve fibers. Using a GFAP-based knock-out of NTE/PNPLA6, we detected an incomplete ensheathment of Remak fibers whereas myelination did not appear to be affected. These results suggest that NTE/PNPLA6 is involved in the maturation of nonmyelinating Schwann cells during development and de-/remyelination after neuronal injury. Since Schwann cells play an important role in maintaining axonal viability and function, it is therefore likely that changes in Schwann cells contribute to the locomotory deficits and neuropathy observed in patients carrying mutations in NTE.

Effects of Neural Crest-Derived Multipotent Stem Cells on Regeneration of an Injured Peripheral Nerve in Mice

We studied the effect of transplantation of neural crest-derived multipotent stem cells (NC-MSCs) obtained from vibrissa germs on regeneration of the injured (transected) sciatic nerve in adult mice (strain FVB). After transplantation of the cells into the injured region, regeneration of the severed nerve was intensified, as compared with that in mice with no implantation. The intensity of vascularization and renewal of the endoneurium also increased. The density of nerve fibers in the distal part of the injured nerve in mice after transplantation of NC-MSCs (10522.8 ± 1044.0 mm–2) was significantly higher than that in mice with nerve injury but without transplantation (8409.5 ± 739.5 mm–2). Possible mechanisms of acceleration of regeneration of the injured peripheral nerve under conditions of transplantation of stem cells are discussed.

Comparación de tres técnicas de trazado retrógrado para la identificación del origen espinal del nervio ciático en ratón.

To identify sensory and motor neurons associated with the sciatic nerve in adult mice, three methods for applying fluorescent tracers (Fluorogold and Dil) were investigated: direct application, intraneural injection and impregnation of a sectioned nerve in a silicone chamber. Most accurate localization of the neurons on the dorsal root ganglia and spinal cord was accomplished by introducing the proximal stump of a transected sciatic nerve into a silicone chamber, filled with tracers and then decalcifying the tissue. Fluorogold was an effective tracing agent, in contrast to Dil, which was not. In addition to associations with cephalic ganglia L4, L5 and L6, as seen in rats, contributory neurons to the sciatic nerve were located in other ganglia in the mouse. These findings show that the silicone chamber-tissue decalcification technique is a viable tool for obtaining comparative neuroanatomical information in the mouse model.

Characterization of a mouse tet-on glia precursor cell line in vitro and in vivo using the electrophysiological measurement

We report here a partial characterization of a “tet-on” glia O2A precursor cell line established from the reverse tetracycline-transactivator (rtTA)-SV40 T antigen (Tag) double transgenic mice. In culture, withdrawal of doxycycline prevents proliferation and the cell line undergoes apoptosis. Importantly, differentiation into type-2-astrocytes and oligodendrocytes can be induced when the cell line is cultured, in the absence of doxycycline, and with epithelial stem cell lines secreting hIL3 or hIL6. In contrast, no maturation into progeny was observed when a hCNTF-secreting cell line was used as the co-culture partner under the same condition. In order to address the question of whether the morphological distinct cells—spindle and stellar shaped cells are of a similar or different cell types, we have performed cell size analysis of these cells by FACS and electro-physiology measurement by the patch clamping technique. They are of a similar cell size, but posses distinct electrophysiological properties—spindle cells are less mature than the stellar cells. These tet-on glia O2A precursor cells were implanted to sites of transected sciatic nerve of adult mice and kept in the precursor stage by feeding mice with doxycycline containing drinking water. The toe movement of injured foot was measured every 3 weeks and the electrophysiological property of motor neuron was determined three months after the operation. Preliminary data have shown that these tet-on glia precursor cells are not toxic to the implanted hosts and can enhance the recovery of damaged motor nerves.

Overcoming Cellular Immunity to Prolong Adenoviral-Mediated Gene Expression in Sciatic Nerve.

In a previous report, we demonstrated that a first generation (E1- and E3-deleted) recombinant adenovirus can transduce expression of the E. coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1. Adenoviral-mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral gene expression over time. In our current work we have optimized adenoviral-mediated gene expression after intraneural injection into sciatic nerve. Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2-week-old animals, decreases thereafter, and there is minimal associated tissue injury. In contrast, very few adenoviral-infected Schwann cells are found in nerves of adult animals 10 days after injection, probably due to immune clearance of viral-infected cells. Consistent with this notion, high levels of lacZ are found in sciatic nerve 30 days after injection of adult SCID mice, which have a genetic defect in both cellular and humoral immunity, of adult β2 microglobulin-deficient mice (β2 M -/-), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenoviral-infected Schwann cells co-cultured with axons in vitro, in the absence of a host immune response, ensheath axons and express lacZ for at least 8 weeks. These data thus demonstrate that expression of first generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell-mediated immunity. Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.

Improved survival of injured sciatic nerve Schwann cells in mice lacking the p75 receptor

Following nerve injury, there is a dramatic increase in the expression of the p75 neurotrophin receptor on Schwann cells (Heumann, R., Korsching, S., Bandtlow, C. and Thoenen, H., Changes of nerve growth factor synthesis in non-neuronal cells in response to sciatic nerve transection. J. Cell Biol., 104 (1987) 1623-1631. Taniuchi, M., Clark, H.B., Schweitzer, J.B. and Johnson, E.M., Expression of nerve growth factor by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties. J. Neurosci., 8 (1988) 664-681.), however the role of the p75 receptor following injury remains unclear. Previous studies have shown that the p75 receptor may play a role in the apoptosis of several cell types. To better understand the role of the p75 receptor in the events following nerve injury, we have compared apoptosis in injured sciatic nerves of adult mice lacking functional p75 receptors and Balb-C (wild-type) mice. Following sciatic nerve crush or resection injuries, we used a fluorescent FragEL DNA fragmentation method to examine the extent of cellular apoptosis in distal nerve segments 5 days, 21 days and 4 months later. Nerve injury induced large numbers of apoptotic nuclei in nerves of both strains, but in p75 knockout mice, the density of apoptotic cells was lower compared to Balb-C mice, 21 days following injury. The p75 receptor may promote apoptosis in Schwann cells when axons are regenerating into the denervated nerve stump.

Insulin-like growth factor binding protein-1 is pre-synaptic at mouse neuromuscular synapses and is transported in nerve.

In a previous study, we localized insulin-like growth factor binding protein 1 (IGFBP-1) to mouse neuromuscular junctions, and intramuscular nerves. To determine if pre-synaptic accumulation of IGFBP-1 occurred, we used double ligation of sciatic nerve in adult mice at different time points. IGFBPs were detected by Western ligand blot (WLB) with 125I-IGF-I. WLB and Western immunoblot (WIB) analysis of extracts from double-ligated nerves showed a delayed (6 days) increase of IGFBP-1 in the soluble fraction between the ligatures and distal to the distal ligature. For comparison we evaluated transport of neurofilament components, using WIB and confirmed the primarily anterograde transport of these intraaxonal proteins. These data suggest that expression of IGFBP-1 is both by activated Schwann cells as well as retrograde axonal transport with likely entry into the axon at the synapse.

Increased rate of peripheral nerve regeneration using bioresorbable nerve guides and a laminin-containing gel

The sciatic nerve of adult mice was transected and proximal and distal nerve stumps were sutured into a nontoxic bioresorbable nerve guide. Nerve guide lumens were either empty or filled with a gel containing 80% laminin and additional extracellular matrix components. Two weeks later cells in the L3 through L5 dorsal root ganglia and the ventral horn of the spinal cord were retrogradely filled with horseradish peroxidase. All animals with the laminin-containing gel but none with empty nerve guides displayed labeled cells. This suggests that the laminin-containing gel significantly hastened axonal regeneration in vivo.

The effects of multiple sequential episodes of demyelination in the sciatic nerve of the mouse.

Intraneural injections of lysophosphatidyl choline were used to examine the effects of multiple episodes of demyelination and repair in the sciatic nerves of adult mice. It was found that the early phases of repair, from initiation of myelinolysis to the appearance of promyelinated fibres, were achieved more rapidly in multiple-injected nerves than in single-injected nerves. However, this lead was temporary, since subsequent remyelination was delayed in the multiple-injected nerves until several days after it had been established in most single-demyelinated nerves. Cell-mediated demyelination was seen in multiple-injected nerves, but never in single-injected nerves. The results are discussed in terms of a the contribution of supernumerary Schwann cells to the acute stage of the programme of cellular repair; and b the possibility that the 'normal' response to an injection of LPC in a nerve which has previously undergone several episodes of demyelination is exacerbated by a local and self-limiting immune-mediated reaction.

Expression of glial antigens C1 and M1 in the peripheral nervous system during development and regeneration

The expression of C1 and M1 antigens was studied by indirect immunofluorescence methods in histological sections of peripheral nerves and ganglia of C57BL/6J mice during development and regeneration. In sciatic nerves of adult mice, C1 but not M1 antigen is found in vimentin- and glial fibrillary acidic protein (GFAP)-positive Schwann cells. A similar distribution is also seen in trigeminal nerve, dorsal root and superior cervical ganglia, and olfactory nerve. In all cases vimentin-positive structures outnumber GFAP- or C1 antigen-positive ones. At birth, C1 antigen and vimentin are expressed in sciatic nerves, but GFAP is not yet detectable. M1 antigen cannot be detected in Schwann cells. In monolayer cultures of neonatal mouse dorsal root ganglia, C1 antigen is expressed in a fibrillary staining pattern in some, but not all morphologically identified Schwann cells. In vitro, M1 antigen is not detectable in Schwann cells. After lesioning sciatic nerves of adult mice by cut or crush, detectable levels of C1 antigen rise after 4–6 days: The number of immunofluorescently labeled structures and their relative intensities are drastically augmented, first distally more so than proximally, over control values from non-lesioned, i.e. contralateral nerves. A similar augmentation is also observed for vimentin and GFAP. M1 antigen expression does not reach detectable levels in Schwann cells under these conditions. The increased detectability of C1 antigen persists up to 150 days after lesioning, the longest time period tested.

The response of the (myelinating) Schwann cell population to multiple episodes of demyelination.

The effects of multiple intraneural injections of the known demyelinating agent lysophosphatidyl choline, LPC, have been studied in the sciatic nerves of adult mice. Up to six separate episodes of demyelination were induced at the same site within the tibial fascicle. Demyelination and subsequent remyelination occurred after each injection. The number of Schwann cells produced during each repair programme exceeded the number required for remyelination and some of the surplus cells temporarily remained associated with the remyelinating axon/Schwann cell units as supernumerary Schwann cells. There was a progressive decline in the proportion of promyelinated axons as the number of injections of LPC increased. The proposition that this could have reflected a more rapid repair following demyelination of thinly (re)myelinated axons was investigated in double LPC-injected nerves. No structures resembling onion bulbs characteristic of hypertrophic neuropathies in man were seen during the course of this study.

Axon Outgrowth Enhanced by a Previous Nerve Injury

In the sciatic nerve of adult mice, the rate of axonal elongation following nerve excision was 27% faster than normal if the nerve had been crushed two weeks before the excision.

The perineurium as a diffusion barrier to protein tracers. Differences between mature and immature animals.

A study was made on the permeability of the sciatic nerve sheaths to various protein tracers in mature and immature animals. Albumin labelled with a fluorescent marker and injected around the sciatic nerve ofadult mice and rats did not penetrate the perineurium to reach the endoneurium. Insuckling mice and rats, the same tracer penetrated the perineurium and spread in the endoneurium of the sciatic nerve. A similar difference in permeability was found when horseradish peroxidase was used as a protein tracer. Newborn guinea pigs, on the other hand, showed a fully developed barrier function of the nerve sheaths to proteins. The difference in permeability between mature and immature animals was also present when tested 2 hours post mortem, demonstrating that the transfer of tracer into the endoneurium of immature animals is not dependent on an oxygen-consuming active process. It has previously been shown that in adult mice a) the perineurium is the structure to which the barrier function of the nerve sheaths is linked and b) the presence of tight junctions between the perineurial cells prevent intercellular diffusion of protein tracers. Ultrastructural observations showed that gaps are present between the cells forming the perineurial lamellae in the sciatic nerve of newborn mice. Horseradish peroxidase passed through these gaps into the endoneurium when injected around the nerve. The difference in permeability of the nerve sheaths between mature and immature mice is therefore presumably due to differences in the apposition of the perineural cells providing open pathways in the young but not in the adult mice.