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PubMed2003

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.

Animals↗
PubMed1999

Anatomic basis of vascularised nerve grafts: the blood supply of peripheral nerves.

The present study was carried out on 30 cadavers (5 fresh, 20 preserved adult and 5 fresh stillborn) following injection of red latex through the subclavian and common iliac arteries. The blood supply to the peripheral nerves was studied in general, together with the vascular pedicles to the ulnar, saphenous, sural, deep and superficial peroneal nerves, and the superficial branch of the radial nerve. The nutrient arteries supplying the peripheral nerves came from either the adjacent axial artery or the fasciocutaneous or muscular arteries. They formed anastomotic channels in the epineurium and penetrated it to form a continuous longitudinal artery. Based on the presence of absence of dominant arterial pedicles, five patterns of blood-supply to the nerves could be identified. I: no dominant arterial pedicle; II: only one dominant artery (e.g. artery with a diameter more than 0.8 mm and accompanying the nerve for most of its length); III: only one dominant vessel that divided into ascending and descending branches to supply the nerve; IV: multiple dominant pedicles; V: multiple dominant arterial pedicles forming a continuous artery that accompanied the nerve. The arterial pedicles to the ulnar, saphenous and deep peroneal nerves and the superficial branch of the radial n. had mean diameters of over 0.8 mm, thus being suitable for microvascular anastomosis. Those to the sural nerve were not present in two thirds of the dissected cadavers. In 10% of the cadavers the superficial peroneal nerve had an arterial pedicle that accompanied the nerve for less than two cm with a mean diameter less than 0.8 mm. The ulnar nerve could be very suitable as a donor vascularized nerve graft as it had a dominant vascular pedicle in all the cases studied; however, its use should be restricted to C8 and T1 root damage of the brachial plexus. The superficial branch of the radial n. might be suitable for vascularized nerve grafting, but this is difficult in practice since the radial artery is a major limb artery. The saphenous nerve had a dominant arterial pedicles in all the cadavers dissected and could be the most suitable as a donor vascularized nerve graft, unlike the sural nerve which did not have a dominant arterial pedicle in two-thirds of the specimens. The deep and superficial peroneal nerves may also be unsuitable since the former is accompanied by a major limb vessel while the latter had a dominant vascular pedicle that accompanied the nerve for only a short distance in 10% of the dissected cadavers.

Aged↗
PubMed2003

Will it be possible to produce peripheral nerves?

Several hundred thousand peripheral nerve injuries occur each year in Europe and the United States alone, mainly as a result of trauma to the upper extremity. Even after optimal surgical repair, functional outcome--especially sensory recovery--is disappointingly poor. This poor outcome is largely due to death of primary sensory neurons, but also lack of fiber regeneration over the nerve trauma zone and target-organ denervation. The type of nerve repair performed is dependent on the size of the nerve gap between the proximal and distal stumps. Short gaps can be repaired directly by end-to-end coaptation and epineural suturing, whereas long nerve gaps require additional material to bridge the defect, which further reduces the functional outcome. The current repair method to bridge nerve defects is the use of autologous nerve grafts (autografts), which provide the regenerating axons with a natural guidance channel populated with functioning Schwann cells (SC) surrounded by their basal lamina. The first use of nerve grafts in humans was reported in 1878, but the wide use of this technique was developed during World War II when nerve grafting became the standard method for nerve-gap repair. Harvesting of nerve grafts results in co-morbidity that includes scarring, loss of sensation, and possible formation of painful neuroma. The donor nerves often are of small calibre and limited number. As functional recovery in peripheral nerve reconstruction is poor, clearly, alternative method for bridging nerve gaps is needed.

Animals↗
PubMed1986

Transmission and scanning electron microscopy of peripheral nerves.

Electron microscopy of peripheral nerve tissue by SEM and TEM facilitates a better understanding of the complex nerve architecture described by early light microscopists. Details of axonal morphology and the Schwann cell/myelin sheath complex in healthy and diseased nerves have been described in the literature. The techniques of tissue harvesting and processing and the observed appearance of peripheral nerves by electron microscopy should be useful to clinicians and researchers involved in peripheral nerve surgery and research.

Animals↗
PubMed1996

Influence of physical parameters of nerve chambers on peripheral nerve regeneration and reinnervation.

We compared reinnervation of target organs after sciatic nerve section leaving gaps of 2, 4, 6, or 8 mm or gaps repaired with silicone tubes in different groups of mice. Functional reinnervation was assessed by noninvasive methods to determine recovery of sweating, nociceptive, and muscular functions in the hindpaw repeatedly during 3 months postoperation. The increase of gap length between nerve stumps delayed the beginning and reduced the degree of functional recovery achieved either with or without repair. When lesions were left unrepaired, functional reinnervation was only noticeable with a 2-mm gap and practically absent with longer gaps. With tube repair, reinnervation started earlier and achieved higher values than in the corresponding unrepaired groups. Tubulization was most effective with 4-mm gaps and comparatively less with shorter and longer gaps. With 4-mm gaps, recovery was higher when the silicone tube had a cross-sectional area 2.5 times that of the sciatic nerve than with narrower or wider tubes and when the wall was the thinnest available. In all cases muscle reinnervation showed a lower progression than sweating and nociceptive recovery.

Action Potentials↗
PubMed1999

Magnetically aligned collagen gel filling a collagen nerve guide improves peripheral nerve regeneration.

Bioresorbable collagen nerve guides filled with either magnetically aligned type I collagen gel or control collagen gel were implanted into 4- or 6-mm surgical gaps created in the sciatic nerve of mice and explanted 30 and 60 days postoperation (dpo) for histological and immunohistochemical evaluation. The hypothesis was that contact guidance of regenerating axons and/or invading nonneuronal cells to the longitudinally aligned collagen fibrils would improve nerve regeneration. The criterion for regeneration was observation of regenerating myelinated fibers distal to the nerve guide. Consistent with previous studies showing poor regeneration in 6-mm gaps at 60 dpo with entubulation repair, only one of six mice exhibited regeneration with control collagen gel. In contrast, four of four mice exhibited regeneration with magnetically aligned collagen gel, including the appearance of nerve fascicle formation. The numbers of myelinated fibers were less than the uninjured nerve in all groups, however, which may have been due to rapid resorption of the nerve guides. An attempt to increase the stability of the collagen gel, and thereby the directional information presented by the aligned collagen fibrils, by crosslinking the collagen with ribose before implantation proved detrimental for regeneration.

Animals↗
PubMed1990

Optic nerve regeneration by peripheral nerve transplant.

We studied the morphology of regenerated retinal ganglion cells and their axons in adult rodents after axotomy and autologous transplantation of the sciatic nerve. Regenerated ganglion cells, backlabeled with rhodamine dextran, were of similar size to or larger than those of intact cells in control animals. Dendrites and occasionally axons as well showed abnormal morphologies in most cells, though some cells appeared quite normal. Cross-sections of the regenerated axons, observed by electron microscopy, were always attached to either the Schwann cell cytoplasm or the basal lamina. The immunoreactive structures to anti-laminin antibody were quite irregular in the cross-sectioned graft and, compared with those of the intact sciatic nerve, they were generally smaller. Their appearance closely resembled that of the basal lamina in the graft observed by electron microscopy. These observations, taken together, suggest that the laminin-rich basal laminae of Schwann cells are essentially important for the regeneration of retinal axons in adult rodents.

Animals↗
PubMed2004

Effects of motor versus sensory nerve grafts on peripheral nerve regeneration.

Autologous nerve grafting is the current standard of care for nerve injuries resulting in a nerve gap. This treatment requires the use of sensory grafts to reconstruct motor defects, but the consequences of mismatches between graft and native nerve are unknown. Motor pathways have been shown to preferentially support motoneuron regeneration. Functional outcome of motor nerve reconstruction depends on the magnitude, rate, and precision of end organ reinnervation. This study examined the role of pathway type on regeneration across a mixed nerve defect. Thirty-six Lewis rats underwent tibial nerve transection and received isogeneic motor, sensory or mixed nerve grafts. Histomorphometry of the regenerating nerves at 3 weeks demonstrated robust nerve regeneration through both motor and mixed nerve grafts. In contrast, poor nerve regeneration was seen through sensory nerve grafts, with significantly decreased nerve fiber count, percent nerve, and nerve density when compared with mixed and motor groups (P < 0.05). These data suggest that use of motor or mixed nerve grafts, rather than sensory nerve grafts, will optimize regeneration across mixed nerve defects.

Animals↗
PubMed2002

Automated and representative fascicle selection for computer-assisted morphometry of myelinated nerve fibres in peripheral nerves.

We describe an algorithm that permits representative fascicle selection for sample acquisition from the entire cross-section of a nerve. We calculate the number of fascicles necessary for the acquisition of a given sample volume of nerve fibres from the image magnification, determine the scheme of intrafascicular sampling, and measure the (estimated) nerve fibre density. We start by storing the positions of all fascicles as coordinates of the microscope motor stage. We then recruit the needed fascicles from the file by calling them up out of the storage order with a constant interval, using the quotient of the total number of fascicles and the number of fascicles necessary to obtain the sample size for this purpose. The results obtained on the analysis of 40 specimens of sural and tibial nerves by means of the image analysis system IBAS are reported.

Algorithms↗
PubMed1999

Highly permeable polylactide-caprolactone nerve guides enhance peripheral nerve regeneration through long gaps.

We compared regeneration and functional reinnervation after sciatic nerve resection and tubulization repair with bioresorbable guides of poly(L-lactide-co-epsilon-caprolactone) (PLC) and permanent guides of polysulfone (POS) with different degrees of permeability, leaving a 6 mm gap in different groups of mice. Functional reinnervation was assessed to determine recovery of motor, sensory and sweating functions in the hindpaw during four months postoperation. Highly permeable PLC guides allowed for faster and higher levels of reinnervation for the four functions tested than impermeable or low-permeable PLC guides, while semipermeable 30 and 100 kDa POS tubes yielded very low levels of reinnervation. The regeneration success rate was higher with PLC than with POS tubes. Morphometrical analysis of cross-sectional nerves under light microscopy showed the highest number of regenerated myelinated fibers at mid tube and distal nerve in high-permeable PLC guides. Impermeable PLC guides allowed slightly worse levels of regeneration, while low-permeable PLC guides promoted neuroma and limited distal regeneration. The lowest number of regenerated fibers were found in POS tubes. In summary, highly permeable bioresorbable PLC guides offer a suitable alternative for repairing long gaps in injured nerves, approaching the success of autologous nerve grafts.

Absorbable Implants↗
PubMed2004

Interposed autologous nerve segment stimulates nerve regeneration in peripheral nerve allografts in a rat model.

The ability of autologous nerve segments interposed between allografts, to increase the total nerve-gap distance, was studied. Sciatic nerve allografts were carried out in a rat model. A 15-mm nerve gap was repaired with a 25-mm nerve graft (interposed group: allo-auto-allograft; control group: allo-allo-allograft). Cyclosporin was given for 12 weeks. Nerve regeneration was evaluated using the weight of the anterior tibial muscle and histologic, morphometric and immunohistochemical analyses at 12, 13, 14, 15, 16, 20, and 24 weeks. Nerve regeneration in the interposed group was statistically significantly better than that in the control group. The authors concluded that a nerve allograft with interposed autograft may enhance nerve regeneration in this model, because of the migration of host-derived Schwann cells into the graft from not only the proximal and distal host nerve stumps, but also the interposed autograft.

Animals↗
PubMedDate not supplied

Connective tissues in the optic nerve and in peripheral nerves: ultrastructural observations.

The connective tissues of the bovine optic nerve and left recurrent nerve have been investigated at the ultrastructure level by TEM and freeze-fracture. The outermost of three concentric sheaths enveloping the nerve appear very similar; the intermediate sheaths appear different enough to suggest different functionalities, while the innermost sheaths are completely dissimilar. The ultrastructural aspects of the extracellular matrix suggest that all the connective tissues may mainly play a passive role as a packing material for the nerve fibers, while none of the sheaths seems apt to withstand mechanical stresses.

Animals↗
PubMed2006

Surgery of the peripheral nerves.

OUTCOME: In contrast to the CNS, peripheral nerves have the ability to regenerate. This ability has been utilized for a long time in the therapy of injuries of peripheral nerves. The aim of the present research was to assess the achievements in reconstructive surgery including nerve grafting and peripheral nerve suture repair in our clinic. MATERIAL AND METHODS: During the 16-year period from 1986 to 2001 we surgically treated 116 patients with peripheral nerve lesions. The cases of lesions of the brachial plexus, partial peripheral nerve lesions and postoperative revision, as well as the attempts of reconstruction of cervical nerves and neurolysis are not included in this work. We analysed the results of surgery in our patients who where subjected to treatment of peripheral nerves in the arms (95 patients) and legs (21 patients). In 53 patients reconstructive surgery was performed by suture repair of peripheral nerve, which included treatment of 65 nerves, 10 patients were treated by suture repair of 2 nerves and repair of 3 nerves in one patient. In 42 patients we reconstructed 45 peripheral nerves of the hand by autograft. The total number of 42 patients compromised 37 males and five females. The average age was 36 years. Double nerve reconstruction (ulnar and median) was accomplished in three patients. In the remaining 21 patients (14 males and seven females) we performed 21 reconstruction operations of 24 injured nerves in the lower extremities, of which 8 operations of 9 nerves involved suture repair of peripheral nerves and 13 operations of 15 nerves were performed using a nerve graft. Their average age was 39.7 years. The average period between primary treatment and operation was 6.7 months. In five cases the interval exceeded 12 months. Operations were performed by microtechnique. All patients were subject to several postoperational tests aimed at the estimation of the degree of recovery in sensory and motor adaptation. The evaluation was performed according the Seddon classification (1975). Motor recovery was evaluated according to a five-point scale and the degree of sensory recovery according to a four-point scale. Recovery of grade M4 and S3 (or higher) was considered an excellent or very good result and grades M3 and S2 were considered a good result. Results corresponding to grades M0-M2 and S0-S1 were considered to be insufficient. RESULTS AND CONCLUSION: The results of reconstruction operations of peripheral nerves injuries were dependent on the patient's age, the period between the injury and operation, the length of the autograft, the location of the injury, the type of injured nerve and the character of the injury (Fig. 2, Tab. 7, Ref: 15).

Adult↗
PubMed1990

Lesion-induced synthesis and secretion of proteins by nonneuronal cells resident in frog peripheral nerve.

Transection of a peripheral nerve results in Wallerian degeneration of the nerve segment distal to the lesion site and the initiation of axonal regeneration just proximal to it (neuroma site). Nonneuronal cells resident in peripheral nerve are suggested to play an important role in neural repair mechanisms through diffusable molecules that they synthesize and secrete. We examined the array of proteins synthesized and secreted by nonneuronal cells resident in the frog peripheral nerve, which is known for its high regenerative capacity. Nerve segments were incubated in medium containing [35S]methionine, and the secreted radioactively labeled proteins were analyzed by gel electrophoresis. Nerve injury resulted in the complete down-regulation of a group of proteins synthesized and secreted by nonneuronal cells in intact nerve. At the same time, the synthesis and secretion of several proteins were up-regulated in the neuroma and degenerating nerve segments, proximal and distal to the axotomy site, respectively. Proteins secreted by the proximal segment were of apparent kDa/pI (mass/isoelectric point) values of 215/5.6, 76/6.7, 73/7.0, 44/5.2, 36.5/5.6, 35.5/6.0, and 32/6.0. Similar proteins were secreted by the degenerating distal segment but with the exception of variable reductions in the 44- and 32-kDa proteins and increases in proteins of apparent kDa/pI values of 39/5.2 and 29/7.3-7.4. Step gradient ultracentrifugation suggested that the latter two are apolipoproteins. Comparison with plasma apolipoproteins further indicated that nerve and plasma apolipoproteins differ. The up-regulation of the synthesis and secretion of these proteins concurrently with nerve degeneration and regeneration strongly imply that these molecules are involved in neuronal repair mechanisms.

Animals↗
PubMed1997

Exogenous opioids influence the microcirculation of injured peripheral nerves.

Local microvessels of peripheral nerve trunks (vasa nervorum) dilate following capsaicin-induced inflammation or local nerve trunk injury. In previous work, we observed that morphine blocked capsaicin-induced dilation of vasa nervorum presumably through the action of local opioid receptors. In the present work, we studied injury-related hyperemia of the rat sciatic vasa nervorum using laser Doppler and hydrogen clearance microelectrode measurements of local perfusion. Systemic morphine reversed hyperemia by vasoconstricting both extrinsic and intrinsic microvessels supplying 48-h-old "neuroma" preparations or crush zones of peripheral nerve trunks. Morphine did not constrict microvessels of contralateral uninjured or sham exposed but uninjured sciatic nerves. In contrast to the injured nerves, contralateral uninjured nerves exposed to morphine frequently had a rise in local perfusion, indicating vasodilation. The vasoconstrictive actions of morphine were blocked by pretreatment with naloxone and were not mimicked by saline injections alone. Systemic doses of selective opioid agonists to mu-, kappa-, and delta-receptors also selectively constricted microvessels of injured nerves. Local blood flow in older experimental neuromas at 7 days had partial sensitivity to morphine, whereas at 14 days perfusion flow was not influenced by morphine. Exogenous opioids dampen early but not later inflammatory microvasodilation and could have important influences on the nerve regenerative milieu.

Animals↗