Adult Peripheral Nerve Research Articles

Peripheral nerve avulsion injuries as experimental models for adult motoneuron degeneration

We have used adult rat peripheral nerve avulsion models to evaluate the effects of neuroprotective molecules on motoneuron degeneration. The right facial nerves of adult Fischer 344 male rats were avulsed and adenoviral vectors encoding glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), transforming growth factor-beta2 (TGFbeta2), and growth inhibitory factor (GIF) were injected into the facial canal. The treatment with the vectors significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase (ChAT) immunoreactivity and suppressed the induction of nitric oxide synthase activity in these neurons. In separate experiments, animals were orally administered a solution of a neuroprotective compound T-588 after avulsion. Both free oral administration and oral tube administration of T-588 improved the survival of injured motoneurons and ameliorated their ChAT immunoreactivity. These results indicate that the gene transfer of GDNF, BDNF, TGFbeta2, and GIF and oral administration of T-588 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Thyroid Hormone Deiodinases in the Central and Peripheral Nervous System

Thyroid hormones play a critical role in development and functioning of the nervous system. Deiodinases (type 2 [D2] and type 3 [D3]) contribute to the control of thyroid hormone action in the nervous system by regulating the local concentrations of triiodothyronine (T(3)), the main active thyroid hormone. Most brain T(3) is indeed locally formed by deiodination of thyroxine (T(4)). This reaction is catalyzed by D2 expressed in astrocytes throughout the brain and in tanycytes in the mediobasal hypothalamus. D3, which inactivates both T(4) and T(3), is mainly expressed in neurons also throughout the brain, with high expression in hippocampus and pyriform cortex. The regulation of deiodinases by many factors in addition to the thyroid hormones indicate that their role is not limited to mitigate the fluctuations in plasma T(4) and T(3). In contrast to the brain, deiodinases are not expressed in the adult peripheral nerve. Nerve lesions induce D2 in peripheral nerve sheaths and D3 in the endoneurial compartment containing Schwann cells. On the basis of available data summarized in this review, D2 and D3 clearly contribute to determine T(3) concentrations depending on the area of the nervous system, the state of development, and the pathophysiologic conditions.

Delayed repair of corticospinal tract lesions as an assay for the effectiveness of transplantation of Schwann cells

We have previously shown that cultured adult olfactory ensheathing cells injected after 8 weeks into functionally complete unilateral lesions of the rat corticospinal tract induce restoration of paw reaching function to about 50% of normal, starting at around 10 days after transplantation. This provides an assay for determining the effectiveness of different methods of cell preparation or different cell types. We report that transplantation of cultured adult peripheral nerve Schwann cells also restores function, but the effect is delayed until around 30 days after transplantation and reaches only around 5-10% of normal. The presence of fibroblasts in the Schwann cell cultures neither improves, nor impairs the reparative effect, but fibroblasts alone (without Schwann cells) have no reparative effect. Without transplantation of exogenous Schwann cells, the ingrowth of endogenous Schwann cells which occurs spontaneously into these lesions does not restore function.

Efficient myelin repair in the macaque spinal cord by autologous grafts of Schwann cells

Experimental transplantation in rodent models of CNS demyelination has led to the idea that Schwann cells may be candidates for cell therapy in human myelin diseases. Here we investigated the ability of Schwann cells autografts to generate myelin in the demyelinated monkey spinal cord. We report that monkey Schwann cells derived from adult peripheral nerve biopsies retain, after growth factor expansion and transduction with a lentiviral vector encoding green fluorescent protein, the ability to differentiate in vitro into promyelinating cells. When transplanted in the demyelinated nude mouse spinal cord, they promoted functional and anatomical repair of the lesions (n = 12). Furthermore, we obtained evidence by immunohistochemistry (n = 2) and electron microscopy (n = 4) that autologous transplantation of expanded monkey Schwann cells in acute lesions of the monkey spinal cord results in the repair of large areas of demyelination; up to 55% of the axons were remyelinated by donor Schwann cells, the remaining ones being remyelinated by oligodendrocytes. Autologous grafts of Schwann cells may thus be of therapeutic value for myelin repair in the adult CNS.

Tight junction proteins ZO-1, occludin, and claudins in developing and adult human perineurium.

In peripheral nerves, groups of Schwann cell-axon units are isolated from the adjacent tissues by the perineurium, which creates a diffusion barrier responsible for the maintenance of endoneurial homeostasis. The perineurium is formed by concentric layers of overlapping, polygonal perineurial cells that form tight junctions at their interdigitating cell borders. In this study, employing indirect immunofluorescence and immunoelectron microscopy, we demonstrate that claudin-1 and -3, ZO-1, and occludin, but not claudin-2, -4, and -5, are expressed in the perineurium of adult human peripheral nerve. We also describe the expression of occludin, ZO-1, claudin-1, -3, and -5 in the developing human perineurium, showing that the expressions of claudin-1 and -3, ZO-1, and occludin follow similar spatial developmental expression patterns but follow different timetables in achieving their respective adult distributions. Specifically, claudin-1 is already largely restricted to perineurium-derived structures at 11 fetal weeks, whereas claudin-3 and occludin are weakly expressed in the perineurial structures at this age and acquire a well-defined perineurial distribution only between 22 and 35 fetal weeks. ZO-1 appears to acquire its mature profile even later during the third trimester. The results of the present and previous studies show that the perineurial diffusion barrier matures relatively late during human peripheral nerve development.

Complement factors in adult peripheral nerve: a potential role in energy metabolism

Complement factors in adult peripheral nerve: a potential role in energy metabolism

Complement factors in adult peripheral nerve: a potential role in energy metabolism

Complement cascade factors are known to play a critical role in myelin clearance after peripheral nerve injury. Here we show that components of both the classical ( C1qa, C1qb, C1qc, C2 and C4) and alternative ( C3, B and adipsin) pathways are expressed by uninjured peripheral nerve as well. mRNAs of components of the alternative pathway were predominantly found in the peri/epineurium, although factor C3 and factor B were also detected in the endoneurial compartment of adult nerve. Interestingly, adipsin mRNA was detected only in peri/epineurium, while adipsin protein was present in both peri/epineurium and endoneurium. This suggests that adipsin is transported to the endoneurium via the circulation from the peri/epineurium or outside of the nerve. Factor 5 and factor 9, necessary for the formation of the membrane–attack complex, were not detected in any part of the healthy peripheral nerve, which together with the observed presence of negative regulators of complement activation, is likely to prevent damage to the healthy nerve caused by complement activation. By analogy with the known role of complement factors in fat, we propose that local expression of these factors plays a role in the regulation of fatty acid homeostasis in the nerve and, thereby, in energy metabolism cross-talk between different compartments of the peripheral nerve.

Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve

The intrinsic capacity of Schwann cells to promote regeneration after limited peripheral nerve lesions has been successfully transferred to extensive peripheral nerve injuries and central nervous system lesions by autologous transplantation strategies. However, both the intrinsic ability of axotomized neurons to regenerate and the permissiveness of the parenchyma surrounding the acute injury site diminish over time. Therefore, the autologous transplantation mode requires a fast and effective method to isolate Schwann cells from peripheral nerve biopsies. Here, we report a method to purify p75 low affinity nerve growth factor receptor (p75LNGFr) expressing Schwann cells from peripheral nerve biopsies in adult rats using magnetic-activated cell separation (MACS). After 1 week of nerve degeneration in culture, nerve fragments were dissociated resulting in mixed cultures containing Schwann cells and fibroblasts. After incubation with specific anti-p75LNGFr antibodies and secondary magnetic bead conjugated antibodies followed by one cycle of MACS, 95% pure Schwann cell cultures were generated as confirmed by flow-cytometry and immunocytochemistry. In contrast to established methods, MACS separation of p75LNGFr expressing cells allows the reliable purification of Schwann cells within 9 days after biopsy employing direct selection of Schwann cells rather than fibroblast depletion assays. Therefore, this method represents an effective and fast means to generate autologous Schwann cells for clinical transplantation strategies aiming for axon repair and remyelination.

Properties of medial gastrocnemius motor units and muscle fibers reinnervated by embryonic ventral spinal cord cells

Severe muscle atrophy occurs after complete denervation. Here, Embryonic Day 14–15 ventral spinal cord cells were transplanted into the distal tibial nerve stump of adult female Fischer rats to provide a source of neurons for muscle reinnervation. Our aim was to characterize the properties of the reinnervated motor units and muscle fibers. Some reinnervated motor units contracted spontaneously. Electrical stimulation of the transplants at increasing intensity produced an average (± SE) of 7 ± 1 electromyographic and force steps. Each signal increment represented the excitation of another motor unit. These reinnervated units exerted an average force of 12.0 ± 1.5 mN, strength similar to that of control fatigue-resistant units. Repeated transplant stimulation depleted 17% of the muscle fibers of glycogen, an indication of some functional reinnervation. Reinnervated (glycogen-depleted), denervated (no cells transplanted), and control fibers were of histochemical type I, IIA, or IIB. Fibers of the same type were grouped after reinnervation. The proportion of fiber types also changed. Reinnervated fibers were primarily type IIA, whereas most fibers in denervated and control muscles were type IIB. Reinnervated fibers of each type had significantly larger cross-sectional areas than the corresponding fiber types in denervated muscles. These data suggest that neurons with different properties can reside in the unusual environment of the adult rat peripheral nerve, make functional connections with muscle, specify muscle fiber type, and reduce the amount that each type atrophies.

Therapeutic efficacy of sonic hedgehog protein in experimental diabetic neuropathy

Hedgehog proteins modulate development and patterning of the embryonic nervous system. As expression of desert hedgehog and the hedgehog receptor, patched-1, persist in the postnatal and adult peripheral nerves, the hedgehog pathway may have a role in maturation and maintenance of the peripheral nervous system in normal and disease states. We measured desert hedgehog expression in the peripheral nerve of maturing diabetic rats and found that diabetes caused a significant reduction in desert hedgehog mRNA. Treating diabetic rats with a sonic hedgehog–IgG fusion protein fully restored motor- and sensory-nerve conduction velocities and maintained the axonal caliber of large myelinated fibers. Diabetes-induced deficits in retrograde transport of nerve growth factor and sciatic-nerve levels of calcitonin gene–related product and neuropeptide Y were also ameliorated by treatment with the sonic hedgehog–IgG fusion protein, as was thermal hypoalgesia in the paw. These studies implicate disruption of normal hedgehog function in the etiology of diabetes-induced peripheral-nerve dysfunction and indicate that delivery of exogenous hedgehog proteins may have therapeutic potential for the treatment of diabetic neuropathy.

Therapeutic efficacy of sonic hedgehog protein in experimental diabetic neuropathy

Hedgehog proteins modulate development and patterning of the embryonic nervous system. As expression of desert hedgehog and the hedgehog receptor, patched-1, persist in the postnatal and adult peripheral nerves, the hedgehog pathway may have a role in maturation and maintenance of the peripheral nervous system in normal and disease states. We measured desert hedgehog expression in the peripheral nerve of maturing diabetic rats and found that diabetes caused a significant reduction in desert hedgehog mRNA. Treating diabetic rats with a sonic hedgehog-IgG fusion protein fully restored motor- and sensory-nerve conduction velocities and maintained the axonal caliber of large myelinated fibers. Diabetes-induced deficits in retrograde transport of nerve growth factor and sciatic-nerve levels of calcitonin gene-related product and neuropeptide Y were also ameliorated by treatment with the sonic hedgehog-IgG fusion protein, as was thermal hypoalgesia in the paw. These studies implicate disruption of normal hedgehog function in the etiology of diabetes-induced peripheral-nerve dysfunction and indicate that delivery of exogenous hedgehog proteins may have therapeutic potential for the treatment of diabetic neuropathy.

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.

A novel technique to isolate adult Schwann cells for an artificial nerve conduit

The use of an artificial nerve conduit containing viable Schwann cells (SCs) is one of the most promising approaches to repair nerve injuries. Obtaining a large number of viable SCs in a short period is demanded for the clinical use of this technique. However, the previous methods using mitogens are not clinically acceptable, and other methods that do not require mitogens, failed to isolate adult SCs effectively or required a long period of time. In this study, we have developed a novel technique to isolate SCs from adult rat peripheral nerves for an artificial nerve conduit without mitogens, which has produced a total number of 1.21×10 5 cells per mg, with an average purity of 93.0±0.58% at 21 days in vitro. The Bottenstein–Sato (BS) medium used in this study, had originally been developed for oligodendrocyte culture, but here it is shown to have an effect on SC proliferation and survival. By changing fetal bovine serum (FBS) concentrations from 0 to 10% serially, SCs could be isolated maximally from the predegenerated nerves while suppressing fibroblast overgrowth. The combination of this technique and the altered medium promoted the migration and proliferation of SCs selectively by utilizing the supporting cells of SCs instead of discarding them by changing the culture dishes and media.

1P-0232 Apolipoprotein A-I-mediated secretion of apolipoprotein E has structural requirements distinct from those related to stimulation of cholesterol efflux

Our previous work demonstrated that the sterol response element binding proteins (SREBP)-1 and SREBP-2, which are the key regulators of storage lipid and cholesterol metabolism respectively, are highly expressed in Schwann cells of adult peripheral nerves. In order to evaluate the role of Schwann cell SREBPs in myelination and functioning of peripheral nerves we have determined their expression during development, after fasting and refeeding, and in a rodent model of diabetes. Our results show that SREBP-1c and SREBP-2, unlike SREBP-1a, are the major forms of SREBPs present in peripheral nerves. The expression profile of SREBP-2 follows the expression of genes involved in cholesterol biosynthesis, while SREBP-1c is co-expressed with genes involved in storage lipid metabolism. In addition, the expression of SREBP-1c in the endoneurial compartment of peripheral nerves depends on nutritional status and is disturbed in type 1 diabetes. In line with this, insulin elevates the expression of SREBP-1c in primary cultured Schwann cells by activating the SREBP-1c promoter. Taken together, these findings reveal that SREBP-1c expression in Schwann cells responds to metabolic stimuli including insulin and that this response is affected in type 1 diabetes mellitus. This suggests that disturbed SREBP-1c regulated lipid metabolism may contribute to the pathophysiology of diabetic peripheral neuropathy.

Mammalian Achaete Scute Homolog 2 Is Expressed in the Adult Sciatic Nerve and Regulates the Expression of Krox24, Mob-1, CXCR4, and p57kip2 in Schwann Cells

The molecular control mechanisms and regulatory molecules involved in nerve repair are not yet well known. Schwann cells have been attributed an important role in peripheral nerve regeneration; therefore, attention has been drawn to regulatory factors expressed by these glial cells. Here, we demonstrate that Mash2, a basic helix-loop-helix (bHLH) transcription factor previously shown to be crucial for placenta development, is expressed by Schwann cells of adult peripheral nerves. We observed that this gene is downregulated after nerve lesion and, using cDNA array hybridization technology, we could demonstrate that Mash2 is a regulator of Krox24, Mob-1, and CXCR4 expression in cultured Schwann cells. In addition, we provide strong evidence that Mash2 is a negative regulator of Schwann cell proliferation. Mash2 represents a first candidate for the missing class B bHLH proteins in peripheral nerves.

Rafts in adult peripheral nerve myelin contain major structural myelin proteins and myelin and lymphocyte protein (MAL) and CD59 as specific markers.

The myelin and lymphocyte protein (MAL) proteolipid is localized in central and peripheral compact myelin membranes, as well as in apical membranes of particular polarized cells. In this study, we addressed the question whether MAL and other peripheral myelin proteins are sorted and targeted to myelin membranes using mechanisms similar to those observed in polarized epithelial cells. To investigate the presence of raft-mediated sorting pathways in Schwann cells, we have isolated and analysed their composition in myelin membranes. Here, we show that rafts are present in adult human and rat peripheral compact myelin membranes and contain MAL, the GPI-anchored protein CD59, and substantial amounts of the PMP22 and P0. Colocalization studies show that CD59, and MAL have an almost identical expression pattern within compact myelin. Moreover, immuno-electron microscopy revealed that MAL, besides its localization in compact myelin, is also localized to Schmidt-Lanterman incisures. Taken together, our results demonstrate the presence of detergent-insoluble glycolipid-enriched complexes (DIGs) in different compartments of myelin membranes and indicate an important role for DIG-mediated transport mechanisms in the maintenance of the adult myelin sheath.

Intrathecal administration of nerve growth factor delays GAP 43 expression and early phase regeneration of adult rat peripheral nerve

Whether nerve growth factor (NGF) promotes peripheral nerve regeneration in vivo, in particular in adults, is controversial. We therefore examined the effect of exogenous NGF on nerve regeneration and the expression of GAP 43 (growth-associated protein 43) in adult rats. NGF was infused intrathecally via an osmotic mini-pump, while control rats received artificial cerebrospinal fluid. Two days after the infusion was initiated, the right sciatic nerves were transected or crushed, and the animals allowed to survive for 3 to 11 days. The right DRG, the right proximal stump of the transected sciatic nerve, and the posterior horn of the spinal cord were examined by Western blotting, immunohistochemistry, and electron microscopy. GAP 43 immunoreactivity in the NGF-treated animals was significantly lower than in the aCSF-treated controls. Electron microscopy showed that the number of myelinated and unmyelinated axons decreased significantly in the NGF-treated rats as compared with the controls. These findings are indicative that exogenous NGF delayed GAP 43 induction and the early phase of peripheral nerve regeneration and supports the hypothesis that the loss of NGF supply from peripheral targets via retrograde transport caused by axotomy serves as a signal for DRG neurons to invoke regenerative responses. NGF administered intrathecally may delay the neurons’ perception of the reduction of the endogenous NGF, causing a delay in conversion of DRG neurons from the normal physiological condition to regrowth state.

Muscle Reinnervation with Delayed or Immediate Transplant of Embryonic Ventral Spinal Cord Cells into Adult Rat Peripheral Nerve

Muscle denervation is common in various neuromuscular diseases and after trauma. It induces skeletal muscle atrophy. Only muscle reinnervation leads to functional recovery. In previous studies, denervated adult rat muscles were rescued by transplantation of embryonic day 14-15 (E14-15) ventral spinal cord cells into a nearby peripheral nerve. In the present study, changes were made in the environment into which the cells were placed to test whether reinnervation was improved by: 1) prior nerve degeneration, induced by sciatic nerve transection 1 week before cell transplantation; 2) transplantation of 1 million versus 5 million cells; 3) addition of nerve growth factor (NGF) to the transplant. Ten weeks after cell transplantation, axons had grown from all of the transplants. The numbers of myelinated axons that regenerated into the tibial, medial (MG), and lateral gastrocnemius-soleus (LGS) nerves were similar across treatments. The mean diameters of large LGS axons (>6 microm) were significantly larger with nerve degeneration before transplantation. The mean diameters of MG and LGS axons were significantly larger with transplantation of 1 million versus 5 million cells. Silver-stained experimental and control lateral gastronemius (LG) muscles showed axons that terminated at motor end plates. Nodal and terminal sprouts were more common in reinnervated muscles (45-63% of all end plates) than in control muscles (10%). Electrical stimulation of the transplants induced weak contractions in 39 of 47 MG muscles (83%) and 33 of 46 LG muscles (72%) but at higher voltages than needed to excite control muscles. The threshold for MG contraction was lower with transplantation of 1 million cells, while LG thresholds were lower without NGF. The cross-sectional area of whole LG muscles was significantly larger with cell transplantation (immediate or delayed) than with media alone, but all of these muscle areas were reduced significantly compared with control muscle areas. These data suggest that delayed transplantation of fewer cells without NGF assists regeneration of larger diameter axons and prevents some muscle atrophy.

Characterization of human nerve basal lamina for their binding properties of anti-MAG antibodies.

The functional importance of the basal lamina in Schwann cell development and in adult peripheral nerve fibers is well known. We have demonstrated previously by confocal microscopy that IgM deposits are present on the basal lamina of myelinating Schwann cells of nerve biopsies from patients with an anti-MAG IgM neuropathy. Therefore, the basal lamina was postulated to represent an early target for the uptake of autoantibodies on the surface of myelinated nerve fibers. In this study, the preparation of cell- and myelin-free basal lamina from human peripheral nerves, using a detergent-dependent method is described and characterized by immunohistochemical and biochemical analysis. Using these methods we demonstrated that an enrichment of basal lamina components of Schwann cells with extraction of myelin could be achieved. Western blot analysis and immunohistochemical characterization showed that anti-MAG IgM antibodies did not recognize an epitope on the basal lamina of normal nerves. The established method will allow in situ investigations of basal lamina components from human peripheral nerves in health and in disease, e.g. peripheral neuropathies of infectious or inflammatory origin.

Schwann cell-enriched cultures from adult human peripheral nerve: a technique combining short enzymatic dissociation and treatment with cytosine arabinoside (Ara-C)

Attempts to design the nerve cellular prostheses have focused on the production of autologous Schwann cells expanded in vitro as the essential component in the regeneration process of injured peripheral nerves . To obtain human Schwann cells of high quality we tested a short enzymatic dissociation protocol that optimized cellular viability levels. We also assessed patterns of bromodeoxyuridine (BrdU) incorporation in both Schwann cells and fibroblasts in the presence or absence of the antimitotic Ara-C, an enrichment option for adult human Schwann cell cultures. The Ara-C treated cultures showed a significantly higher Schwann cell percentage (95%), compared with that obtained in the absence of Ara-C (70%), indicating that this antimitotic acts to eliminate fibroblasts in each one of the applied pulses (four pulses). However, we have observed that the use of this antimitotic during prolonged periods of time produced a cumulative effect causing Schwann cell cytotoxicity. Therefore, we consider that our enzymatic dissociation technique and the application of only two pulses of Ara-C to the cultures are enough to achieve enrichment of adult human Schwann cells in culture.