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Purpose: To compare nerve regeneration in autologous detergent extracted and freeze-thawed muscle grafts and to electrophoretically characterize the grafts. Methods: Autologous acellular muscie grafts were created either by freeze/thawing or by detergent extraction and then used to bridge a 10 mm gap in rat sciatic nerve. The autologous grafts were compared with respect to protein content, using electrophoresis preimplantation, and axonal outgrowth, Schwann cell and macrophage content, using immunocytochemistry (neurofilaments, S-100 protein, ED 1 macrophages) at 5–20 days postimplantation. Results: The extracted muscle grafts were elastic, but the amount of several proteins was reduced and laminin was still present at a position of basal laminae of the muscle fibers. The freeze/thawed grafts were brittle and lacked elasticity, but resulted in minor changes in major proteins. The axons regenerated through both types of grafts (initial delay 6 days and rate 0.7-0.8 mm/day), which shrunk in length by 25%. There were no apparent differences with respect to Schwann cells and macrophages. Conclusions: The results suggest that detergent extracted muscle tissue, in which some basal lamina proteins remain but cells are removed, could present a new favourable option for nerve grafting. [ABSTRACT FROM AUTHOR]

We demonstrate high-fidelity guidance of axons using rows of nanowires. The axons are prevented from crossing the rows, making it possible to guide and sort a large number of axons as opposed to when chemical patterns are used. Focal adhesion forms at the nanowires establishing a possible site of information transfer between the surface and the cells. Rows of gallium phosphide (GaP) nanowires were epitaxially grown on GaP(111) substrates in patterns defined by electron beam lithography.

xonal outgrowth on smooth and porous silicon surfaces was studied in organ culture. The pore size of the silicon substrata varied between 100 and 1500 nm. We found that axons preferred to grow and elongate on porous silicon surfaces only when pores of (150-500 nm) are available.

Dissociated sensory neurons were cultured on epitaxial gallium phosphide (GaP) nanowires grown vertically from a gallium phosphide surface. Substrates covered by 2.5 microm long, 50 nm wide nanowires supported cell adhesion and axonal outgrowth. Cell survival was better on nanowire substrates than on planar control substrates. The cells interacted closely with the nanostructures, and cells penetrated by hundreds of wires were observed as well as wire bending due to forces exerted by the cells.

Activation of extracellular-signal-regulated kinase-1/2 (Erk1/2) by phosphorylation to p-Erk1/2, and proliferation of Schwann cells were investigated in the rat sciatic nerve by immunohistochemistry. Axotomy in vivo and culturing of nerve segments in vitro resulted in a rapid (30 min) increase of p-Erk1/2 in Schwann cells with peaks at 2 and 24 h. Proliferation measured by bromodeoxy uridine incorporation and immunostaining in vivo and in vitro 48 h after axotomy showed an increase in Schwann cell proliferation at the sites of Erk1/2 activation. The Erk1/2 inhibitor U0126 inhibited both the increase in p-Erk1/2 and the bromodeoxy uridine incorporation. We suggest that an increase in p-Erk1/2 is required for nerve injury-induced proliferation of Schwann cells.

The aim of this study was to investigate if costimulation blockade could be used to modulate the immune response, to prevent rejection, and to stimulate regeneration into nerve allografts. Nerve allografts from Balb/C mice, and isogenic nerve grafts (isografts) from C57/BL6 mice, were used to bridge a 7-mm gap of the sciatic nerve in C57/BL6 mice. Allograft recipients were treated with either a triple treatment with anti-lymphocyte function antigen-1 (anti-LFA), anti-CD40 ligand (anti-CD40L), and cytotoxic T-lymphocyte antigen 4 immunoglobulin (anti-CTLA4Ig) or isotype antibodies (placebo) at postoperative days 0, 2, 4, and 6 (intraperitoneal). After 5 or 9 days, the nerve grafts, together with the proximal and the distal nerve segments, were evaluated by histology and immunocytochemistry for inflammatory cells [CD4-positive (CD4+) and CD8-positive (CD8+) staining cells] and axonal outgrowth (neurofilaments). The immune response was inhibited by costimulation blockade with less extensive inflammation and a lower number of CD4+ staining cells in triple-treated allografts at 9 days. The regeneration rate was significantly faster in isografts (0.75 mm/day) compared with allografts with placebo treatment (0.39 mm/day), but not when compared with triple-treated allografts (0.49 mm/day). At 9 days, the axons were significantly longer in nerve isografts than in nerve allografts, irrespective of treatment. Hence, costimulation blockade neither increased the regeneration rate nor the outgrowth length in triple-treated allografts. We conclude that costimulation blockade inhibits the immune response in nerve allografts without deterring early axonal outgrowth.

The production of galectin-3, a carbohydrate-binding mammalian lectin, is upregulated in Schwann cells after peripheral nerve injury in areas where Schwann cells proliferate. Here we tested if galectin-3 affected proliferation of Schwann cells in cultured sciatic nerve segments. Galectin-3 significantly decreased the number of bromodeoxyuridine-labelled Schwann cell nuclei. Neither lactose nor a synthetic inhibitor directed against the carbohydrate-binding region abolished the effects of galectin-3. In addition, a mutant galectin-3 unable to bind endogenous carbohydrates had similar effects as normal galectin-3. We conclude that galectin-3 reduces proliferation of Schwann cells in cultured sciatic nerve segments by a mechanism which is independent of its carbohydrate-binding moiety.

Calpain, a Ca2+-dependent cysteine protease, has been implicated in neuronal apoptosis following spinal cord injury. In this study, activation of calpain was investigated in motor neurons of adult spinal cord slices from the mouse, using a cell-permeable fluorogenic calpain substrate and Western blotting. Calpain was rapidly activated in the motor neurons of excised spinal cord slices and calpain activity was observed both in the cytoplasm and the nuclei. In these neurons, nuclear and chromatin condensation were pronounced. Both calpain inhibitor VI and EGTA (ethyleneglycol-bis(beta-aminoethyl ether) N' ,N' ,N' ,N' -tetraacetic acid) inhibited calpain activation and subsequent appearance of apoptotic nuclei. In contrast, the general caspase inhibitor Z-VAD.fmk had no effect. Calpain activation was also observed in the slices by Western blotting using an antibody to 150-kD calpain-cleaved alpha-fodrin fragment. These results show that calpain is rapidly activated in injured motor neurons and imply that this activation could be responsible for execution of caspase-independent apoptosis in injured adult motor neurons.

In this study, we developed a microdispenser technique in order to create protein patterns for guidance of neurites from cultured adult mouse dorsal root ganglia (DRG). The microdispenser is a micromachined silicon device that ejects 100 picolitre droplets and has the ability to position the droplets with a precision of 6-8 microm. Laminin and bovine serum albumin (BSA) was used to create adhesive and non-adhesive protein lines on polystyrene surfaces (cell culture dishes). Whole-mounted DRGs were then positioned close to the patterns and neurite outgrowth was monitored. The neurites preferred to grow on laminin lines as compared to the unpatterned plastic. When patterns were made from BSA the neurites preferred to grow in between the lines on the unpatterned plastic surface. We conclude that microdispensing can be used for guidance of sensory neurites. The advantages of microdispensing is that it is fast, flexible, allows deposition of different protein concentrations and enables patterning on delicate surfaces due to its non-contact mode of operation. It is conceivable that microdispensing can be utilized for the creation of protein patterns for guiding neurites to obtain in vitro neural networks, in tissue engineering or rapid screening for guiding proteins.

Staining by activating transcription factor 3 (ATF3), a neuronal marker of nerve injury, was examined by immunocytochemistry in neurons and Schwann cells after crush or transection (regeneration inhibited) of rat sciatic nerve. ATF3 immunoreactivity peaked in neurons after three days and then gradually subsided to normal within 12 weeks after the crush. The response lasted somewhat longer and declined over time in spinal cord neurons but not in those of dorsal root ganglia (DRG) after transection, indicating a differential regulation of sensory and motor neurons. ATF3 expression was more pronounced in Schwann cells, and remained longer after transection, implying that to some extent regenerating axons produce signals that reduce ATF3 expression in Schwann cells. However, even after transection without repair (no contact with regenerating axons), ATF3 expression in Schwann cells in the distal segment decreased over time suggesting that regenerating axons are not entirely responsible for the down-regulation. These findings have clinical implications on when it is worthwhile to reconstruct nerve injuries.

Here, we investigated the effect of calpain inhibitors on apoptosis in organotypic adult spinal cord slices from mice. An increase in calpain I immunoreactivity was found in the nuclei of motor neurons from slices cultured for 30 min. After 4 h, the immunopositive motor neurons exhibited apoptotic changes including nuclear and chromatin condensation. Eight hours after excision, most motor neurons showed nuclear apoptotic features. Two calpain inhibitors, leupeptin and calpain inhibitor XI, inhibited apoptosis in the motor neurons while the caspase inhibitor Z-VAD.fmk had no effect. Leupeptin, but not calpain inhibitor XI and Z-VAD.fmk, also inhibited nucleosomal DNA fragmentation. These results suggest the involvement of calpain I in the induction of apoptosis in motor neurons of adult spinal cord and that apoptosis can be triggered independent of caspase activation.

Nanotechnology has provided methods to fabricate surface patterns with features down to a few nm. If cells or cell processes exhibit contact guidance in response to such small patterns is an interesting question and could be pertinent for many applications. In the present study we investigated if axonal outgrowth was affected by nano-printed patterns in polymethylmethacrylate (PMMA)-covered silicon chips. To this end adult mouse sympathetic and sensory ganglia were mounted in Matrigel on the chips close to the nano-patterns. The patterns consisted of parallel grooves with depths of 300 nm and varying widths of 100-400 nm. The distance between two adjacent grooves was 100-1600 nm. The chips were cultured in medium containing 25 ng/ml of nerve growth factor to stimulate axonal outgrowth. After 1 week of incubation, axonal outgrowth was investigated by immunocytochemistry or scanning electron microscopy. Axons displayed contact guidance on all patterns. Furthermore, we found that the nerve cell processes preferred to grow on ridge edges and elevations in the patterns rather than in grooves, a seemingly claustrophobic behavior. We conclude that axons of peripheral neurons might be guided by nanopatterns on PMMA when the lateral features are 100 nm or larger. The present results can be utilized for nerve regenerating scaffolds or the construction of a stable, high-resolution electronic interface to neurons, which is required for future brain machine interfaces.

Regeneration occurs if a distal nerve segment is attached end-to-side to an intact donor nerve after the nerve has been injured. We investigated if attachment of a proximal nerve segment as well, as an extra source of axons, had any advantage over the single attachment of a distal nerve segment to the same donor nerve. In rats, a single distal radial nerve, or both the proximal and distal radial nerve segments, were attached end-to-side to the musculocutaneous nerve, and effects were examined up to eight months after the repair. Cell profiles were double-labelled, indicating recruitment of axons by collateral sprouting, but there were few such cells. There was a shift in the distribution of retrograde labelled neuronal cell profiles in spinal cord and in DRGs between the two types of repair. Both procedures resulted in axonal outgrowth and some functional recovery, but there was no improvement if a proximal nerve segment was also used as a source for axons.

We wanted to find out if any of three different types of manipulations: a piece of muscle or nerve put parallel to a nerve; an epineurial window made, or sutures inserted into a nerve, or both; or pieces of nerve sutured to an epineurial window end-to-side to the musculocutaneous or sciatic nerve, resulted in activation of activating transcription factor 3 (ATF3) in neurons and in non-neuronal cells. ATF3, a marker of cell activation, was investigated by immunocytochemistry one week after manipulation. A piece of nerve or muscle parallel to a nerve did not induce ATF3 locally in the nerve and induced ATF3 only rarely in neurons. In contrast, an epineurial window or insertion of sutures, or both, with or without attachment of a piece of nerve placed end-to-side, induced robust ATF3 expression. We conclude that an injury to a peripheral nerve trunk associated with end-to-side nerve repair, activates neurons and non-neuronal cells and may contribute to sprouting of axons into the nerve attached end-to-side.

We investigated the functional outcome of c-Jun activation in sympathetic and sensory neurons of neonatal rat superior cervical ganglion (SCG) and dorsal root ganglion (DRG), respectively. Distinctly different roles of c-Jun activation have been suggested for these two types of neurons. In dissociated sympathetic neurons, c-Jun has been demonstrated to promote apoptosis, whereas in sensory neurons it stimulates axonal outgrowth. In organ-cultured ganglia, we found that c-Jun was activated within 24 h of explantation in both types of neurons, and that the JNK inhibitor SP600125 could mitigate this response. In both types of neurons, c-Jun activation was also reduced by NGF treatment. Inhibition of c-Jun activation did not affect the viability of sympathetic neurons, whereas the number of apoptotic sensory neurons increased. Furthermore, inhibition of c-Jun reduced axonal outgrowth from both SCG and DRG. Thus, in organ culture, c-Jun activation may be required for axonal outgrowth and, at least in sensory neurons, it promotes survival. The role of ATF3, a neuronal marker of injury and a c-Jun dimerization partner, was also examined. We found an ATF3 induction in both SCG and DRG neurons, a response, which was reduced by JNK inhibition. The reduction of ATF3 upon JNK inhibition was much larger in DRG than in SCG, a result which might account for the higher number of apoptotic neurons in JNK inhibitor exposed DRG. Taken together, and contrary to our expectations, neonatal sympathetic and sensory neurons seem to respond to axonal injury similarly with respect to c-Jun activation, and in no case was this activation pro-apoptotic.

c-Jun activation has been implicated not only in neuronal apoptosis, but also in survival and regeneration. This Janus facet of c-Jun activation could be related to neuronal cell type or to the developmental stage of the neuron. We investigated c-Jun activation in E18 sensory neurons. Cultures of rat dorsal root ganglia neurons were maintained with or without the addition of nerve growth factor or the c-Jun N-terminal kinase inhibitor, (D)-JNKI1. Few dorsal root ganglia neurons survived nerve growth factor deprivation, whereas neurons supplied with nerve growth factor survived and exhibited extensive axonal outgrowth. Activated c-Jun was present in the nuclei of neurons with regenerating axons, but not in apoptotic neurons. c-Jun N-terminal kinase inhibition reduced the number of p-c-Jun immunoreactive and regenerating neurons, and increased cell death. Thus, activation of c-Jun seems to be required for survival and regeneration of developing sensory neurons.

Motor neuron cell death was studied in organotypic cultures of adult spinal cord slices from the mouse. Six hours after excision, many motor neuron nuclei displayed apoptotic features including nuclear and chromatin condensation. At this time point, many motor neurons also exhibited immunoreactivity to calpain II. Both the calpain inhibitor VI and ethyleneglycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) prevented the appearance of apoptotic nuclei whereas the pan caspase inhibitor Z-VAD.fmk had no effect. The results suggest that calpain is required for apoptosis of motor neurons and that this type of apoptosis is independent of caspase activation.

In the present study, we investigated if the previously observed JNK-mediated activation of c-Jun and induction of ATF3 could be ascribed to axonal transport of JNK signaling components, or if axonal transport of the transcription factors themselves contributes to the nuclear changes in injured sensory neurons. We observed retrograde axonal transport of a number of JNK upstream kinases in ligated rat sciatic nerve. In these preparations, axonal transport of JNK/p-JNK, the JNK scaffolding protein JIP, and the transcription factors ATF3 and ATF2/p-ATF2 was also found. No or little retrograde transport of c-Jun and p-c-Jun was found, whereas an anterograde transport of Hsp27, a protein previously reported in the context of p-c-Jun and ATF3, was observed. In separate experiments, we found that in vitro inhibition of axonal transport or axonal inhibition of JNK reduced the number of p-c-Jun- and ATF3-positive neuronal nuclei. The results suggest that retrograde axonal transport of JNK signaling components contributes to the injury induced c-Jun phosphorylation and ATF3 induction.

To characterize the gene activity that may be required for neuronal survival and regeneration, we used the Affymetrix GeneChip Mu74A to screen 12000 genes and expressed sequence tag (EST) mRNA from L4 and L5 mouse dorsal root ganglia (DRG) 12 h and 24 h after sciatic nerve transection. At 12 h, we found 17 upregulated transcripts, and at 24 h, 49 that met our criteria of a significant 2-fold increase in expression. The alterations included a total of eight transcription factors and several genes associated with TGF-beta- and IL-6-mediated signaling. Two of the changes, amphiregulin and plasminogen activator inhibitor-1 (PAI-1), were confirmed by real-time quantitative PCR (QPCR). Addition of amphiregulin (20 ng/ml) to organ-cultured DRG stimulated axonal outgrowth while PAI-1 (20 nM) inhibited migration of Schwann cells from the ganglia.

The end-to-side nerve-repair technique, i.e., when the distal end of an injured nerve is attached end-to-side to an intact nerve trunk in an attempt to attract nerve fibers by collateral sprouting, has been used clinically. The technique has, however, been questioned. The aim of the present study was to investigate end-to-side repair in the upper extremity of rats with emphasis on functional recovery, source, type, and extent of regenerating fibers. End-to-side repair was used in the upper limb, and the radial or both median/ulnar nerves were attached end-to-side to the musculocutaneous nerve. Pawprints and tetanic muscle force were used to evaluate functional recovery during a 6-month recovery period, and double retrograde labeling was used to detect the source of the regenerated nerve fibers. The pawprints showed that, in end-to-side repair of either one or two recipient nerves, there was a recovery of toe spreading to 60-72% of the preoperative value (lowest value around 47%). Electrical stimulation of the end-to-side attached radial or median/ulnar nerves 6 months after repair resulted in contraction of muscles in the forearm innervated by these nerves (median tetanic muscle force up to 70% of the contralateral side). Retrograde labeling showed that both myelinated (morphometry) sensory and motor axons were recruited to the end-to-side attached nerve and that these axons emerged from the motor and sensory neuronal pool of the brachial plexus. Double retrograde labeling indicated that collateral sprouting was one mechanism by which regeneration occurred. We also found that two recipient nerves could be supported from a single donor nerve. Our results suggest that end-to-side repair may be one alternative to reconstruct a brachial plexus injury when no proximal nerve end is available.

We hypothesised that pieces of nerve that are normally trimmed away during graft repair could be a source of Schwann cells and used to create an autologous cell-containing nerve graft. To test the idea a transsection injury was made on the rat sciatic nerve. Seven days later, pieces from the proximal and distal stumps were used for immediate isolation of its non-neuronal cells. These cells, of which 80% were Schwann cells, were injected into a silicone tube bridging a 10 mm gap on the same nerve. Tubes containing cells showed superior regeneration of nerve fibres across the gap compared with tubes with no cells. This new repair technique is rapid, does not require the sacrifice of a healthy donor nerve, or the time-consuming culturing and expansion of Schwann cells. The method has the potential to replace current methods for repair of extended nerve injuries.

Nuclear translocation of activating transcription factor 3 (ATF3), used as a marker for neuronal injury, was studied by immunocytochemistry in sensory neurons in dorsal root ganglia and locally along the sciatic nerve after severe chronic nerve compression in rats. In compressed nerves (application of a narrow silicone tube), ATF3 immunoreactivity was seen in the nucleus of sensory neurons and in Schwann cells below the compression at two and four weeks. Removal of the silicone tube (decompression) at two weeks did not affect the number of ATF3-positive cell nuclei in dorsal root ganglia. No ATF3 immunoreactivity was found in undamaged nerves on the other side. Functional variables (toe spreading and muscle force) were impaired at two weeks, with partial improvement at four weeks. Nerve compression induces nuclear translocation of ATF3, a transcription factor associated with survival and regeneration of sensory neurons. The response is related to duration of compression and partly correlated to function.

The role of c-Jun activation for survival and regeneration of sensory neurons is unclear. Here we report that c-Jun N-terminal kinase (JNK)-mediated c-Jun activation is important for axonal outgrowth of sensory neurons in rat nodose and dorsal root ganglia (DRG). Peripheral severance of the vagus or the sciatic nerve resulted in a massive and rapid, but transient increase of the activated JNK (p-JNK) in neuronal nuclei, followed by c-Jun phosphorylation and activating transcription factor-3 (ATF3) induction. JNK inhibition by the selective JNK inhibitors SP600125 and (D)-JNKI1 did not affect neuronal survival in explanted or dissociated ganglia, but dramatically reduced axonal outgrowth, c-Jun activation, and ATF3 induction. Using retrograde labeling, we demonstrated that activated c-Jun (p-c-Jun) and ATF3 were associated with regenerative neurons. Taken together, our results suggest that JNK-mediated c-Jun activation is one of the first cell body reactions in response to nerve injury and that this activation and subsequent ATF3 induction are associated with axonal outgrowth.

The aim was to establish an accurate, reproducible, and simple method to evaluate functional recovery after different types of nerve injuries to the brachial plexus of rats. To that end, pawprints, measured as distance between the first and fourth and second and third digits, were used for evaluation of injuries including crush injury, transection/repair, or graft repair of the median, ulnar, and radial nerves. Immunocytochemistry of the C-terminal flanking peptide of neuropeptide Y (CPON) and neurofilaments was used to investigate the cell body response and axonal outgrowth, respectively. Functional recovery was dependent on the severity as well as on the level of the lesion. Neither a single injury to the median nerve nor an injury to the ulnar nerve affected the pawprint, while an injury to both these nerves or a single injury to the radial nerve caused impairment of pawprints. There was a rapid recovery after crush injury to these nerves compared to previous reports of a similar injury to the sciatic nerve. The pattern of axonal outgrowth was related to the severity of the lesion. A conditioning lesion, i.e., an initial lesion of the same nerve preceding a test injury by a few days, of both motor/sensory fibers led to a quicker functional recovery. Surprisingly, conditioning of only sensory fibers had nearly the same effect. The cell body response was dependent on the level of the nerve lesion. The upper extremity of rats might be useful to evaluate the effects of new repair methods after nerve injuries using functional evaluation with pawprints as a simple and accurate method.

Regeneration of the rat sciatic nerve through acellular muscle and nerve autografts was evaluated 6-28 days postoperatively by the sensory pinch test, immunocytochemical staining for neurofilaments, and light and electron microscopy. Data points generated by the pinch test were plotted against postoperative time periods and by the use of regression analysis the initial delay period for muscle grafts was determined to 10.3 days. This value was similar to that previously published for acellular nerve grafts (9.5 days), but significantly longer than that for fresh nerve grafts (3.6 days). The calculated regeneration rate (slope of the regression line) for muscle grafts (1.8 mm/day) did not differ significantly (p > 0.05) from that calculated for acellular nerve grafts (2.1 mm/day) or for fresh nerve grafts (1.5 mm/day). The front of regenerating axons shown by axonal neurofilament staining confirmed the pinch test results. Both types of acellular grafts were repopulated with host non-neuronal cells and the muscle graft contained occasional ectopic muscle fibres. Remnants of graft basal laminae were evident at the ultrastructural level. These results indicate the suitability of either acellular muscle or nerve grafts for nerve repair despite their prolonged initial delay periods compared with conventional fresh nerve grafts.