Expression of specific sheath cell proteins during peripheral nerve growth and regeneration in mammals

Protein synthesis in the nerve sheath of injured as well as intact mature and developing sciatic nerves from rat and rabbit was investigated by incubating segments of nerve with [35S]methionine in vitro. The composition of labeled proteins under the different conditions of nerve growth was analyzed by two-dimensional gel electrophoresis and fluorography. The expression of six secreted proteins in rat sciatic nerve with the apparent molecular weights of 70,000 (70 kD), 54,000 (54 kD), 51,000 (51 kD), 39,000 (39 kD), 37,000 (37 kD), and 30,000 (30 kD) was of particular interest because of the correlation of their synthesis and secretion with aspects of nerve growth and regeneration. The synthesis of the 37-kD protein was significantly stimulated during both sciatic nerve development as well as regeneration but not in the intact mature nerve. The expression of this protein appears to be regulated by signal(s) from the axon but not the target. The 70-kD protein was exclusively synthesized in response to axotomy, thus confining its role to some aspect(s) of nerve repair. In contrast, the 54- and 51-kD proteins were expressed in the intact mature nerve sheath. Their synthesis and release was rapidly inhibited upon axotomy but returned to normal or higher levels towards the end of sciatic nerve regeneration, suggesting a role in the maintenance of the integrity of the mature (nongrowing) rat nerve. The 39- and 30-kD proteins were only transiently synthesized within the first week after axotomy. Two proteins with the apparent molecular masses of 70 and 37 kD were synthesized in denervated rabbit sciatic nerve. The similar molecular weights, net charges, and time-courses of induction suggest a homology between these proteins in rabbit and rat, indicating common molecular responses of peripheral nerve sheath cells to axon injury in both mammalian species.     

Supramolecular aggregation and organization in peripheral nerve myelin

Under certain preparative conditions the lipid bilayers of glutaraldehyde-fixed, PNS myelin demonstrate a marked compartmentalization, which can be augmented by lipid extraction following sectioning. The results are interpreted as indicating a supramolecular domain pattern of arrangement centered upon the transmembrane protein (P0) molecules. The latter are thought to be surrounded by annuli of substantially immobilized phospholipids. In the lamellar planes particular lipids are considered to have a nonrandom distribution. The visualization of bilayer compartmentalization was seen only in negatively stained sections obtained from unembedded or glutaraldehyde-urea-embedded myelin. Lipids were unextracted in the basic preparations except in so far as some unfixed, amphipathic molecules escaped at the trough-fluid interface at the time of sectioning, an observed phenomenon which probably aided in the visualization of the compartmentalization. Visualization was also augmented by surface tension expanding section fragments as they floated on the trough fluid. All stages of transition between well-ordered myelin and dispersed globular units were commonly to be found. Deliberately delipidated myelin exposed more sharply defined and smaller globular units in bilayer regions, but even these are regarded as being supramolecular aggregates including residual lipid annuli around the transmembrane proteins. The addition of cadmium ions as a "fixative" for lecithin seemed to improve the preservation of glutaraldehyde-urea-embedded myelin but was not strictly necessary to reveal its domain structure. A secondary tannic acid fixation was required to process unembedded myelin so as to reveal the fundamental compartmentalization of its lipid bilayers.     

Fluidity of the myelin sheath in the peripheral nerves of diabetic rats

In the present study we examined the structural integrity of the myelin sheath in the peripheral nerves from short-term streptozotocin (STZ)-treated diabetic rats, using ESR spectroscopy as a tool in determining the dynamic state and the structure of the myelin lipid phase. Experiments were performed on spin-labeled sciatic and sural nerves from STZ-treated Hannover-Wistar rats and age-matched controls. The spectrum analysis employed a numerical simulation model with the set of fitting parameters that in the same time relate the ESR line shape and structure and dynamics of the probed environment. The simulation considered three spectral components weighted and summed in the composite spectrum. The comparative analysis of results showed the fraction of the spectral component II to be significantly increased in the spectra of diabetic rats, indicating the significant increase in overall fluidity of the myelin structure. The origin of fluidity changes was further investigated using an experimental model for demyelination (local injection of ethidium bromide in vivo), proteolytic action of trypsin in vitro, and osmotic myelin swelling in vitro. Analysis and comparison of the results suggested a conclusion in terms of changed biophysical properties of the myelin lipid phase in peripheral nerves in the pathology of diabetes.     

Anti-CD40 ligand antibody permits regeneration through peripheral nerve allografts in a nonhuman primate model

Systemic immunosuppression is typically required to prevent allograft rejection. Antibody-based therapies that induce immune unresponsiveness represent an appealing alternative to nonspecific immunosuppression, which is often associated with significant morbidity. In mice, successful prevention of nerve allograft rejection has been demonstrated through interference with the CD40/CD40 ligand interaction. This study investigated the effectiveness of anti-CD40 ligand monoclonal antibody as single-agent therapy in preventing rejection and supporting nerve regeneration across long nerve allografts in nonhuman primates. Twelve outbred cynomolgus macaques were arranged into six genetically mismatched pairs, with each animal receiving a 5-cm ulnar nerve allograft in the right arm and a 5-cm autograft in the left arm. Mixed lymphocyte reaction assays were used to assess resulting immune unresponsiveness. Treated animals (n = 10) received anti-CD40 ligand monoclonal antibody 10 mg/kg one time, locally applied, and 20 mg/kg systemically on postoperative days 0, 1, 3, 10, 18, and 28, and then monthly. Untreated animals (n = 2) served as the untreated controls. At 4 or 6 months after transplantation, nerves were harvested for histological analysis. Four treated animals underwent an additional challenge after cessation of anti-CD40 ligand monoclonal antibody therapy and nerve graft harvests. Autogenous and allogeneic skin and nerve inlay grafting was performed to assess the permanence of immune unresponsiveness induced by anti-CD40 ligand monoclonal antibody. Animals that received anti-CD40 ligand monoclonal antibody demonstrated robust regeneration across nerve allografts, similar to that seen in the autograft control in the contralateral arm. The histomorphometric analysis of allografts in the untreated animals demonstrated significantly worse measurements compared with their matched autograft controls. Animals that received anti-CD40 ligand monoclonal antibody with concomitant skin allografts had virtually no evidence of nerve regeneration through allografts. Allogeneic skin and nerve allografts applied 2 to 12 months after withdrawal of anti-CD40 ligand monoclonal antibody therapy were consistently rejected. This study demonstrates that anti-CD40 ligand monoclonal antibody prevents rejection and allows regeneration of peripheral nerve allografts in nonhuman primates. The effect of anti-CD40 ligand monoclonal antibody appears to be transient, however, with restoration of immunocompetence shortly after withdrawal of therapy.     

Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1-centimeter nerve gap

A new type of a biodegradable nerve graft conduit material, the amnion tube, has been developed in our laboratory. To test the tube in the peripheral nerve regeneration process, it was initially applied across a 1-cm sciatic nerve gap in rats and was compared with other nerve conduit materials. We used male Sprague-Dawley rats as our animal model. The experiment included 66 rats that were randomly assigned into five groups: autograft (n = 17), amnion tube (n = 19), silicone tube (n = 20), no repair (n = 7), and sham group (n = 3). The process of peripheral nerve regeneration was evaluated at 2, 4, 10, and 17 weeks following injury and repair by using morphologic and functional assessments of the outcome of nerve regeneration in each animal. Nerve regeneration across the amnion tube nerve conduit was comparable with that seen in autograft and superior to that of the silicone group. A uniform nerve tissue was seen filling and crossing the amnion conduit, and the regenerated nerve from the proximal stump reached the distal end and was undifferentiated from the normal nerve tissues. At 4 months, the amnion tube biodegraded and no longer could be identified and differentiated from the nerve tissues. The amnion tube animal group showed a number of axons very close to that in the nerve autograft group (37,157 versus 33,054). Functional recovery at a 2- to 4-week interval was significantly statistically higher only in the amnion tube animal group (p = 0.01). However, the improvement disappeared between 10 and 17 weeks. In conclusion, the amnion tube is a potential ideal nerve conduit material secondary to its unique characteristics: it contains important neurotropic factors, is biodegradable, provokes a very weak immune response, is semiflexible, is readily available, and is easily manufactured into different sizes and diameters.     

Glycosylation of myelin glycoproteins in peripheral nerve via lipid intermediates

Membrane preparations from chick peripheral nervous system (PNS) catalyzed the transfer of [³H]glucose from UDP-[³H]glucose into glucosylphosphoryl dolichol. The initial rate of glucosylphosphoryl dolichol formation in a non-myelin membrane fraction from actively myelinating chick PNS was 11 fold higher than that from adult. Exogenous dolichyl monophosphate stimulated glucosylphosphoryl dolichol synthesis in both fractions. The higher level of glucosylphosphoryl dolichol synthesis corresponded to the onset of myelination in chick PNS. Exogenous dolichyl monophosphate also stimulated the labeling of glucosylated oligosaccharide lipids and glycoproteins in the fraction. On SDS polyacrylamide gel electrophoresis, the relative mobility of the major and minor radioactive glycoprotein corresponded with that of the P0 and PASII glycoprotein in PNS myelin, respectively. The results suggest that myelin glycoproteins in PNS are glycosylated via lipid intermediates.     

Nerve regeneration through side-to-side neurorrhaphy sites in a rat model: a new concept in peripheral nerve surgery

Despite great improvement and refinements in nerve repair techniques, there were still problems in repair of peripheral nerve injuries for which proximal stumps were not available. In these circumstances for which classic end-to-end neurorrhaphy was impossible, new treatment modalities, benefiting by an adjacent healthy nerve, have been under investigation to overcome this problem. Therefore, end-to-side nerve repair with its modifications came to view and axonal passages through this site were shown. Moreover, the results were unsatisfactory or necessitating sacrifice of another healthy nerve. Three groups, containing 10 rats each, were included in the study. First was the control group, with end-to-end repair of the peroneal nerve. Second was the end-to-side repair group, in which the distal stump of the peroneal nerve trunk was anastomosed to the lateral side of the tibial nerve. The third was the side-to-side repair group. In this technique, 1-mm diameter epineural windows, both from peroneal and tibial nerve trunks facing each other, were removed and side-to-side neurorrhaphy was performed. After 3 weeks, as the second step, the peroneal nerve was sectioned proximally. At 2, 4, 8, 12, 20, and 28 weeks, functional assessment of nerve regeneration was performed by using walking track analysis. The number of myelinated fibers and fiber diameters were measured and an electron microscopic evaluation was carried out. Statistically, both in morphometric and gait analysis, the differences in values between the groups were significant in favor of the control group, followed by the side-to-side group. The study showed that axonal passage was possible with side-to-side technique and the functional results were satisfactory and superior to the end-to-side technique. Continuous supply of neurotrophic factors from their target cells was the probable cause of superior functional return in side-to-side repair, because both joining nerves were intact and healthy during the anastomosis procedure and after 3 weeks. It was concluded that this technique could be indicated in salvage of nerves in cases for which any intermediate segments would be removed, as in tumor ablation surgery, harvesting of nerve grafts, or both.     

The peripheral nerve allograft: an assessment of regeneration in the immunosuppressed host

Regeneration across the nerve allograft in the immunosuppressed host was assessed using electrical and histologic parameters. The Lewis rat (RTIl) served as the recipient animal, and ACI rats (RTIa) provided the donor nerve allografts. Hydrocortisone and azathioprine were used in various dose schedules as the immunosuppressive agents. Animals were immunosuppressed for either 30 or 100 days. Histologic and electrophysiologic measurements of nerve regeneration were assessed at 30, 100, and 180 days. The degree of nerve regeneration was similar in all experimental groups. Short-term, low-dose immunosuppression was as successful as longer-term, higher-dose immunosuppression therapy. The degree of nerve regeneration in all experimental groups was significantly better than that in the fresh, untreated nerve allograft control group (Lewis/ACI) but was not as good as that seen in the autograft control group (Lewis/Lewis).     

Localization and irregular distribution of Na,K-ATPase in myelin sheath from rat sciatic nerve

Sodium, potassium adenosine triphosphatase (Na,K-ATPase) is a membrane-bound enzyme that maintains the Na(+) and K(+) gradients used in the nervous system for generation and transmission of bioelectricity. Recently, its activity has also been demonstrated during nerve regeneration. The present study was undertaken to investigate the ultrastructural localization and distribution of Na,K-ATPase in peripheral nerve fibers. Small blocks of the sciatic nerves of male Wistar rats weighing 250-300g were excised, divided into two groups, and incubated with and without substrate, the para-nitrophenyl phosphate (pNPP). The material was processed for transmission electron microscopy, and the ultra-thin sections were examined in a Philips CM 100 electron microscope. The deposits of reaction product were localized mainly on the axolemma, on axoplasmic profiles, and irregularly dispersed on the myelin sheath, but not in the unmyelinated axons. In the axonal membrane, the precipitates were regularly distributed on the cytoplasmic side. These results together with published data warrant further studies for the diagnosis and treatment of neuropathies with compromised Na,K-ATPase activity.     

A mechanical model for collagen fibril load sharing in peripheral nerve of diabetic and nondiabetic rats

Peripheral neuropathy affects approximately 50% of the 15 million Americans with diabetes. It has been suggested that mechanical effects related to collagen glycation are related to the permanence of neuropathy. In the present paper, we develop a model for load transfer in a whole nerve, using a simple pressure vessel approximation, in order to assess the significant of stiffening of the collagenous nerve sheath on endoneurial fluid pressure. We also develop a fibril-scale mechanics model for the nerve, to model the straightening of wavy fibrils, producing the toe region observed in nerve tissue, and also to interrogate the effects of interfibrillar crosslinks on the overall properties of the tissue. Such collagen crosslinking has been implicated in complications in diabetic tissues. Our fibril-scale model uses a two-parameter Weibull model for fibril strength, in combination with statistical parameters describing fibril modulus, angle, wave-amplitude, and volume fraction to capture both toe region and failure region behavior of whole rat sciatic nerve. The extrema of equal and local load-sharing assumptions are used to map potential differences in diabetic and nondiabetic tissues. This work may ultimately be useful in differentiating between the responses of normal and heavily crosslinked tissue.