Distal Stump Of Transected Nerve Research Articles

A two-compartment organotypic model of mammalian peripheral nerve repair

Schwann cells in the distal stump of transected nerve upregulate growth factors that support regeneration on a modality-specific basis. It is unclear, however, which of these preferentially support motor axon regeneration. Identification of these factors will require a model that can isolate growth factor effects to growing axons while reproducing the complex three-dimensional structure of peripheral nerve. A two-compartment PDMS base is topped by a collagen-coated membrane that supports a spinal cord cross-section above one compartment. Fluorescent motoneurons in this section reinnervate a segment of peripheral nerve that directs axons through a water-tight barrier to the second compartment, where nerve repair is performed. Motoneurons remain healthy for several weeks. The axons they project through the water-tight barrier survive transection and cross a nerve repair in substantial numbers to reinnervate an additional nerve segment. Fluidic isolation of the two compartments was confirmed with a dye leakage test, and the physiologic integrity of the system was tested by retrograde labeling of only those motor neurons to which tracer was exposed and by limitation of toxin effects to a single compartment. Nerve repair cannot be modeled in monolayer cell culture. Our previous organotypic model accurately modeled nerve repair, but did not allow individual control of motoneuron and growth cone environments. This model isolates treatment effects to growing axons while reproducing the complex three-dimensional structure of peripheral nerve. Additionally, it facilitates surgical manipulation of tissues and high-resolution imaging.

MR imaging and T2 measurements in peripheral nerve repair with activation of Toll-like receptor 4 of neurotmesis

To investigate the role of MR imaging in neurotmesis combined with surgical repair and Toll-like receptor 4 (TLR4) activation. Forty-eight rats received subepineurial microinjection of the TLR4 agonist lipopolysaccharide (LPS, n = 24) or phosphate buffered saline (PBS, n = 24) immediately after surgical repair of the transected sciatic nerve. Sequential fat-suppressed T2-weighted imaging and quantitative T2 measurements were obtained at 3, 7, 14 and 21 days after surgery, with histologic assessments performed at regular intervals. T2 relaxation times and histological quantification of the distal stumps were measured and compared. The distal stumps of transected nerves treated with LPS or PBS both showed persistent enlargement and hyperintense signal. T2 values of the distal stumps showed a rapid rise to peak level followed by a rapid decline pattern in nerves treated with LPS, while exhibiting a slow rise to peak value followed by a slow decline in nerves treated with PBS. Nerves treated with LPS exhibited more prominent macrophage recruitment, faster myelin debris clearance and more pronounced nerve regeneration. Nerves treated with TLR4 activation had a characteristic pattern of T2 value change over time. Longitudinal T2 measurements can be used to detect the enhanced repair effect associated with TLR4 activation in the surgical repair of neurotmesis. • TLR4 activation had additional beneficial effects on neurotmesis beyond surgical repair. • TLR4 activation had a characteristic time course of T2 values. • T2 measurements can help detect beneficial effects with TLR4 activation.

Dynamic changes of p27(kip1) and Skp2 expression in injured rat sciatic nerve.

S phase kinase-associated protein 2 (Skp2), an F-box protein, is required for the ubiquitination and consequent degradation of p27(kip1). Previous reports have showed that p27(kip1 )played important roles in cell cycle regulation and neurogenesis in the developing central nervous system. But the distribution and function of p27(kip1 )and Skp2 in nervous system lesion and regeneration remains unclear. In this study, we observed that they were expressed mainly in both Schwann cells and axons in adult rat sciatic nerve. Sciatic nerve crush and transection resulted in a significant up-regulation of Skp2 and a down-regulation of p27(kip1). By immunochemistry, we found that in the distal stumps of transected nerve from the end to the edge, the appearance of Skp2 in the edge is coincided with the decrease in p27(kip1) levels. Changes of them were inversely correlated. Results obtained by coimmunoprecipitation and double labeling further showed their interaction in the regenerating process. Thus, these results indicate that p27(kip1 )and Skp2 likely play an important role in peripheral nerve injury and regeneration.

Oxidized galectin-1 stimulates the migration of Schwann cells from both proximal and distal stumps of transected nerves and promotes axonal regeneration after peripheral nerve injury.

Oxidized galectin-1 has recently been identified as a key factor that plays important roles in initial axonal growth in injured peripheral nerves. The aim of this study was to investigate the effects of oxidized galectin-1 on regeneration of rat spinal nerves using acellular autografts (containing no viable cells) and allografts (containing no cell membranes) with special attention to the relationship between axonal regeneration and Schwann cell migration. Immunohistochemically, endogenous galectin-1 was expressed in dorsal root ganglion (DRG) neurons, spinal cord motoneurons, and axons and Schwann cells in normal sciatic nerves. Administration of oxidized recombinant human galectin-1 (rh-gal-lox, 5 ng/ml) in autograft model promoted axonal regeneration from motoneurons as well as from DRG neurons; this was confirmed by a fluorogold tracer study (p < 0.05). Anti-rh-gal-1 antibody (30 microg/ml) strongly inhibited axonal regrowth (p < 0.05). Pretreatment of allografts with rh-gal-lox stimulated the migration of Schwann cells not only from proximal stumps but also from distal stumps into the grafts, resulting in accelerated axonal regeneration (p < 0.05). Moreover, Schwann cell migration preceded the axonal growth in the presence of exogenous rh-gal-lox in the grafts. These results strongly suggest that local administration of exogenous rh-gal-lox promotes the migration of Schwann cells followed by axonal regeneration from both motor and sensory neurons, resulting in acceleration of neuronal repair. This technique may also be of value in the repair of human nerves.

Ultrastructural Study of Anterograde Transport of Glial Cell Line-Derived Neurotrophic Factor from Dorsal Root Ganglion Neurons of Rats towards the Nerve Terminal

The glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic substance in the central and peripheral nervous systems. The present immunohistochemical study clarified the ultrastructural localization of GDNF-immunoreactive substance (GDNF-IR) accumulated at transfected sciatic nerve stumps and also at normal spinal dorsal horn, and has demonstrated that GDNF-IR products appear to be located in dense-cored vesicles within the axons. Furthermore, to determine the source of proximally accumulated GDNF in the transected sciatic nerve, we attempted a transection and a double ligation maneuver involving the sciatic nerve. In the early period after the ligation (20 h), GDNF-IR fibers were observed in the proximal and distal segment of the ligations, but no immunoreactivities were detected in the middle segment. On the other hand, at a late period (8 days) after the transection, GDNF-IR fibers had almost disappeared, but weak GDNF-IR was observed in Schwann cells in the proximal and distal stumps of transected nerve. These findings suggest that most of GDNF-IR was transported from the proximal or distal side in the early period, but was locally synthesized by Schwann cells around the ligations in the late period. Spinal rhizotomy caused prominent accumulation of GDNF-IR products at the cut end of the ganglion side of the dorsal root, but not at the ventral root. These results suggested that dorsal root ganglionic (DRG) sensory neurons are one of the origins of GDNF. The fact that small- to medium-sized DRG neurons show enhanced GDNR-IR after the colchicine treatment may support the above suggestion. In conclusion, the present results strongly suggest that a subgroup of DRG sensory neurons synthesized GDNF-containing dense-cored vesicles in the neuronal somata and anterogradely transports the vesicles to peripheral or central axon terminals.

Differential Patterns of ERK and STAT3 Phosphorylation after Sciatic Nerve Transection in the Rat

Peripheral nerve injury induces a specific pattern of expression of growth factors and cytokines, which regulate injury responses and regeneration. Distinct classes of growth factors and cytokines signal through specific intracellular phosphorylation cascades. For example, the ERK phosphorylation cascade mediates signaling through transmembrane tyrosine kinase receptors and the JAK/STAT cascade mediates signaling through the GP130 receptor complex. We tested whether specific phosphorylation patterns of ERK and STAT3 result from nerve injury and whether such phosphorylation correlates with the expression of specific growth factors and cytokines. At sites adjacent to a nerve transection, we observed that ERK phosphorylation peaked early, persisted throughout 16 days, and was equally intense at proximal and distal sites. In contrast, STAT3 phosphorylation peaked later than ERK but did not persist as long and was stronger in the proximal than in the distal segment adjacent to the injury. In addition, in distal segments further away from the injury site, ERK became phosphorylated with a delayed time course, while STAT3 remained unphosphorylated. These patterns of phosphorylation correlated well with the expression of neurotrophin and interleukin-6 mRNAs in the distal stump. In addition, we found that the pattern of SAPK phosphorylation is similar to the pattern observed for STAT3, while the pattern of macrophage infiltration into the transected nerve was distinct from all the phosphorylation patterns observed. Together, these observations suggest that ERK activation is important in the establishment of a regeneration-promoting extracellular environment in the far distal stump of transected nerves and that STAT3 activation is important in the control of cellular responses close to the site of injury.

Developing Schwann Cells Acquire the Ability to Survive without Axons by Establishing an Autocrine Circuit Involving Insulin-Like Growth Factor, Neurotrophin-3, and Platelet-Derived Growth Factor-BB

Although Schwann cell precursors from early embryonic nerves die in the absence of axonal signals, Schwann cells in older nerves can survive in the absence of axons in the distal stump of transected nerves. This is crucially important, because successful axonal regrowth in a damaged nerve depends on interactions with living Schwann cells in the denervated distal stump. Here we show that Schwann cells acquire the ability to survive without axons by establishing an autocrine survival loop. This mechanism is absent in precursors. We show that insulin-like growth factor, neurotrophin-3, and platelet-derived growth factor-BB are important components of this autocrine survival signal. The secretion of these factors by Schwann cells has significant implications for cellular communication in developing nerves, in view of their known ability to regulate survival and differentiation of other cells including neurons.

Nerve Regeneration in Predegenerated Basal Lamina Graft: The Effect of Duration of Predegeneration on Axonal Extension

We used predegenerated acellular grafts to bridge proximal and distal stumps of transected nerves and studied how the duration of predegeneration might affect axonal regeneration. Predegenerated acellular grafts were prepared by transecting the tibial nerve of donor rats and, after a period of degeneration, freeze-thawing a 40-mm long segment of the distal stump. Five degeneration periods were used: 0 days (for fresh grafts), 3 days, 1 week, 4 weeks, and 8 weeks. Fresh cellular grafts not treated with freeze-thawing were also used for comparison. Each graft was then transplanted to an isogeneic recipient rat, in which it was used to bridge the proximal stump of the transected left tibial nerve and the distal stamp of the transected right tibial nerve. Six weeks were allowed for the regeneration of axons in all grafts. The regeneration was then assessed by studying transverse sections of the grafts, to determine the maximum length that the axons had regenerated, and the packing density of axons (percentage of sampled areas occupied by axons). The results show that axons had grown to the maximum length in the 4-week predegenerated grafts, and had the highest packing density in the 1-week predegenerated grafts. Regeneration in the fresh acellular (0-day predegenerated) and 8-week predegenerated grafts, especially the latter, was poor. We examine the results with reference to time-dependent events of Wallerian degeneration and propose that there are beneficial effects of multiple factors on the grafts during the first 4 weeks of predegeneration, causing a slow but significant improvement in their capability to support axonal growth. The subsequent rapid deterioration of such capability may be related to structural changes in the extracellular scaffold.

Neurofilament and tubulin mRNA expression in Schwann cells.

Feeding of elemental tellurium to weanling rats blocks synthesis of cholesterol (a major component of myelin), and causes demyelination of the sciatic nerve. Expression of mRNA for myelin-specific genes in Schwann cells is downregulated. We now demonstrate specificity for Schwann cell injury in that expression of mRNAs for neurofilament subunits and for class II beta-tubulin (parameters sensitive to axonal injury) is unaltered in neurons of the dorsal root ganglia. An unexpected result was that in tellurium-treated rats there was marked upregulation of expression of mRNAs coding for the light and medium neurofilament subunits ("neuron-specific" proteins) as well as that for class II beta-tubulin (the major neuronal beta-tubulin isotype) in Schwann cells. Expression of these "neuronal" mRNA species was also detected in distal stumps of transected nerves at times when Schwann cells were undergoing dedifferentiation.

04 and A007-sulfatide antibodies bind to embryonic Schwann cells prior to the appearance of galactocerebroside; regulation of the antigen by axon-Schwann cell signals and cyclic AMP

In the rat sciatic nerve, the relationship between Schwann cells, axons, the extracellular matrix and perineurial sheath cells undergoes extensive modification between embryo day 15 and the onset of myelination during the first postnatal day. Little is known about molecular changes in Schwann cells in this important prenatal period. In the present paper, we use immunofluorescence to study the prenatal development and postnatal regulation of the antigen(s) recognized by the 04 monoclonal antibody and a well-characterized rat monoclonal antibody to sulfatide, A007. We show that, in a series of immunochemical tests, the 04 antibody recognizes only sulfatide in neonatal and adult rat nerves. Both antibodies first bind to Schwann cells in the sciatic nerve at embryo day 16-17, and all Schwann cells bind both antibodies at birth. In the adult nerve, both nonmyelin-forming and myelin-forming cells are labelled with the antibodies. Schwann cells dissociated from embryo day 15 nerves and cultured in the absence of axons develop neither 04 nor A007 binding on schedule, and 04-positive and A007-positive Schwann cells from postnatal nerves lose the ability to bind these antibodies during the first few days in culture. Schwann cells in the distal stump of transected nerves also sharply down-regulate cell surface binding of 04. High numbers of 04-positive or A007-positive Schwann cells reappear in cultures treated with agents that mimic or elevate intracellular cAMP. We conclude that two anti-sulfatide antibodies 04 and A007, recognize an antigen, probably sulfatide, that appears very early in Schwann cell development (one to two days prior to galactocerebroside) but is nevertheless subject to upregulation by axonal contact or elevation of intracellular cAMP.

Specificity in regenerative outgrowth and target reinnervation by mammalian peripheral axons

There are indications that specific factors are present in the distal stump of transected nerves which preferentially attract axons of the corresponding proximal stump into the distal nerve stumps. However, the impact of these factors is unclear, since there is abundant evidence that numerous regenerating motor and sensory axons are topographically misdirected after nerve transection and repair. Topographic reinnervation is improved after fascicular repair of fasciculated nerves, and quite precise after nerve crush. The latter may not be true, however, for non-myelinated axons, which show a high degree of aberrant growth even after crush. In contrast, regenerative outgrowth appears to be topographically specific after neonatal nerve transection. Reinnervation of muscle fibers appears to be unspecific in adult mammals, but specific after neonatal injury under certain circumstances. Some preference for reinnervation of the appropriate sensory receptors seems to exist although this preference does not preclude reinnervation of receptors by 'foreign' sensory fibers. In conclusion, incorrect topographic and target reinnervation commonly occurs after peripheral regeneration in adult mammals, and most certainly explains some of the functional disturbances after peripheral nerve lesions. Topographic regeneration appears to be better after nerve injury in developing mammals indicating that mechanisms from the developmental period may persist and aid in accurate regenerative outgrowth.

Immunocytochemical localization of S-100b protein in degenerating and regenerating rat sciatic nerves.

We studied the cellular and subcellular distribution of S-100b protein in normal, crushed, and transected rat sciatic nerves by an immunocytochemical procedure. In uninjured nerves, S-100b protein was restricted to the cytoplasm and membranes of Schwann cells, with no reaction product present in the nucleus or in axons. Similar images were seen from the first to the thirtieth day after the crush in activated Schwann cells during the degeneration period, i.e., up to the seventh post-lesion day, and in normal Schwann cells reappearing during the regeneration period, i.e., after the seventh post-lesion day, in the zone of the crush and proximal and distal to it. By the technique employed, there seemed to be no differences in the intensity of the immune reaction product in normal and activated Schwann cells. Also, similar images were seen in the proximal stump of transected nerves. Only a slight S-100b protein immune reaction product could be observed in the rare activated Schwann cells present in the distal stump around the seventh post-lesion day, the majority of cell types being represented by fibroblasts and elongated cells at this stage and thereafter. By immunochemical assays, similar results as those presented here have been reported and interpreted as indicative of the presence of S-100 protein in axons or, alternatively, of axonal control over expression of S-100 protein in Schwann cells. Our immunocytochemical data clearly show that the strong reduction in the S-100 protein content of the distal stump of transected nerves is owing to the paucity of Schwann cells and to the decrease in the S-100 protein content of these cells, rather than to degeneration of axons.

An in vivo assay of neurotropic activity

Previous studies in this laboratory indicate that diffusible factors from distal stumps of transected peripheral nerves exert a neurotropic effect on regenerating nerve fibers in vivo. The present study was performed (a) to strengthen this hypothesis, and (b) to begin to determine the distribution and identity of putative neurotropic factors in peripheral nerves. Rat sciatic nerves were transected and inserted into the inlet end of a hollow 6 mm-long Y-shaped implant made of medical-grade Silastic tubing. To one of the outlet ends was attached an Elvax pellet impregnated with whole homogenate from a single sciatic nerve. The other outlet was attached to a pellet not containing homogenate. Homogenates assayed were obtained from distal stumps of transected nerves derived 14 days postoperatively or from unoperated sciatic nerves. After 3.5 weeks, nerve fiber bundles consisting of myelinated regenerating axons were only present in implant forks attached to pellets containing denervated nerve homogenate. This activity was heat- and trypsin-sensitive. These data (a) strengthen the hypothesis that diffusible factors from distal nerve stumps of transected nerves can support nerve fiber regeneration, (b) indicate that those factors are protein and/or protein dependent, and (c) suggest that unoperated peripheral nerve (at the levels assayed) either do not contain such factors or contain substances which inhibit such factors.

Tropism in nerve regeneration in vivo. Attraction of regenerating axons by diffusible factors derived from cells in distal nerve stumps of transected peripheral nerves

We re-examined the hypothesis of Cajal 3, later refuted by Weiss and Taylor 20, that cells in distal stumps of transected peripheral nerves exert an attractive (tropic) effect on regenerating axons. This question was re-assessed in vivo using surgical materials and assay procedures not available to those workers. Proximal stumps of transected rat sciatic or cat peroneal nerves were inserted into the single inlet end of a hollow, Y-shaped Silastic implant. Regenerating axons were provided with alternative targets consisting of a vacant arm vs one occupied by a sciatic nerve graft (rat), or a tibial (T out) vs peroneal (P out) distal nerve stump (cat). In some cases P out was rendered metabolically compromised relative to T out by exposing the former to dry ice and inhibitors of DNA and RNA synthesis. At 4.5 or 6 weeks postoperatively, the number of regenerating axons in each fork of the implant was assessed by morphometric analysis (total number of non-myelinated and myelinated axons greater than 1 μm in diameter at 4.5 weeks, and total number of myelinated axons at 6 weeks postoperatively), or by quantification of an axonally transported label. Rat sciatic nerve fibers exclusively regenerated toward the nerve graft, suggesting the existence of a neurotropic lure. In cats, morphometric analysis revealed at 10- (4.5 week) and 6-fold (6 week) greater number of axons growing towards untreated T out vs treated P out. When both distal stumps were untreated, more axons were seen in forks leading to P out. Analysis of transported label confirmed the preferential growth of axons towards untreated T out vs treated P out for both motor and sensory axons. In separate experiments, Nuclepore filters (0.2 μm, pore size) were inserted between distal nerve stumps and outlet ends of Silastic implants. Preferential regeneration toward untreated stumps was observed if the distance between proximal and distal nerve stumps was equal to but not greater than 4–5 mm. These results suggest that peripheral nerve fiber regeneration in vivo can be directed by cells in distal stumps of transected nerves, and that this effect can be mediated over distances of several millimeters via diffiusible factors.

Effect of the length of the distal stump of transected nerve upon the rate of degeneration of taste buds.

The present work was carried out to study the effect of the length of the distal stump of transected nerve upon the rate of degeneration and the time course of disappearance of mammalian taste buds. Twelve adult rabbits were anaesthetized and the glossopharyngeal nerves of both sides were exposed and transected so as to leave a long distal stump on the right and a short one on the left side. The animals were sacrificed at different post-operative periods ranging from 2 to 14 days and the circumvallate and foliate papillae of both sides were examined. The taste buds on the side of the short distal stump always showed a greater decrease in number, size and cell contents than those of the side of the long distal stump. The taste buds on the side of the short distal stump disappeared earlier than those of the side of the long distal stump. Vallate taste buds disappeared earlier than the foliate: possible reasons for this were put forwards. The validity of the neurohumoral theory to the mammalian taste buds was discussed.