Regenerating motor bridge axons refine connections and synapse on lumbar motoneurons to bypass chronic spinal cord injury

To restore motor control after spinal cord injury requires reconnecting the brain with spinal motor circuits below the lesion. A bridge around the injury is an important alternative to promoting axon regeneration through the injury. Previously, we reported a novel motor bridge in rats. The thirteenth thoracic nerve was detached from the muscle it innervates and the cut end implanted caudally into the lumbar gray matter where motor bridge axons regenerate. In this study, we first determined that regenerating bridge axons project to spinal motor circuits. Stable projections were present in ventral motor laminae of the cord, including putative synapses directly on motoneurons, 2 months after insertion in the intact cord. At this time, earlier-forming dorsal horn projections were mostly eliminated. Regenerating axons were effective in evoking leg motor activity as early as 2 weeks. We next determined that bridge axons could regenerate caudal to a chronic injury. We hemisected the spinal cord at L2 and inserted the bridge nerve 1 month later at L5 and found ventral laminae projections similar to those in intact animals, including onto motoneurons directly. Finally, we determined that the bridge circuit could be activated by neural pathways rostral to its origin. For spinally hemisected animals, we electrically stimulated the rostral spinal cord and recorded evoked potentials from the bridge and, in turn, motor responses in the sciatic nerve. Our findings suggests that bridge motoneurons could be used by descending motor pathways as premotor interneurons to transmit neural signals to bypass a chronic spinal injury.     

Histological assessment in peripheral nerve tissue mgineering

The histological analysis of peripheral nerve regeneration is one of the most used methods to demonstrate the success of the regeneration through nerve conduits. Nowadays, it is possible to evaluate different parameters of nerve regeneration by using histological, histochemical, immunohistochemical and ultrastructural techniques. The histochemical methods are very sensible and are useful tools to evaluate the extracellular matrix remodeling and the myelin sheath, but they are poorly specific. In contrast, the immunohistochemical methods are highly specific and are frequently used for the identification of the regenerated axons, Sehwann cells and proteins associated to nerve regeneration or neural linage. The ultrastructural techniques offer the possibility to perform a high resolution morphological and quantitative analysis of the nerve regeneration. However, the use of a single histological method may not be enough to assess the degree of regeneration, and the combination of different histological techniques could be necessary.     

Nerve regeneration through holey silicone tubes

Recent studies focus on regeneration where nerve stumps are placed in a silicone tube. Since the tube is impermeable, the fluid and cells that collect from the stumps bath the axons. This is presumably beneficial. Making the tube permeable by making holes in its walls should change the patterns of regeneration. If this is done, the major cytologic change is an increase in the fascicular perineurium. There are more individual fascicles, more cells line each fascicle and the lining cells are coated by more prominent external laminae than after similar regeneration in a regular silicone tube or in the normal untransected nerve. For axonal numbers, there are more myelinated and unmyelinated axons in the gap and more unmyelinated axons in the distal stump than after regeneration in a regular silicone tube. The numbers in the holey tube regenerate are statistically different from normal but they are closer to normal than after similar regeneration in a regular silicone tube. There are significantly fewer myelinated and unmyelinated axons than in the normal sural nerve after regeneration through a holey tube, but there are more than after regeneration through a regular tube. The numbers of axons in the nerve to the medial gastrocnemius muscle are not significantly different from normal or from the other regeneration paradigms. These data allow the suggestion that regeneration through a silicone tube with macroscopic holes in its walls may be superior in certain respects to regeneration through a regular impermeable silicone tube.     

Contribution of reference electrode to the compound muscle action potential

In compound muscle action potential (CMAP) recording, the contribution by the reference electrode is considered to be much smaller than that of the active electrode. We tested this assumption by making quantitative measurements of the signals recorded individually by the active and reference electrodes. In the thenar (median nerve) and extensor digitorum brevis (peroneal nerve) muscles, the reference electrode did contribute less. In the hypothenar muscle (ulnar nerve), however, the signals recorded by active and reference electrodes were of similar amplitude. In tibial nerve conduction studies (NCS), the CMAP from the abductor hallucis (AH) muscle was recorded mainly by the reference electrode; the large-amplitude signal recorded by the reference electrode is attributed to volume-conducted activity from other muscles stimulated during the study. The onset latency of the potential recorded by the active and reference electrodes was similar despite significantly different distances from the stimulating site. Hence, the merits of using anatomic landmarks for defining the distal stimulation site are assessed. When the reference electrode makes a large contribution, the CMAP amplitude may not decrease commensurate with any wasting of the muscle under the active recording electrode, and the need to use another muscle for recording the CMAP for that nerve should be considered.     

Effect of different optic nerve lesions on retinal ganglion cell death in the frog Rana pipiens

Following optic nerve crush in various species of frog, a proportion of the retinal ganglion cells re-establishes functional contact with the optic tectum. However, as much as 50% of the retinal ganglion cells die during this process. The determinants of an individual ganglion cell's fate have not been established. In this study of Rana pipiens, cell survival after optic nerve crush was compared with that after nerve cut followed by stump separation, a procedure that considerably delayed entry of optic axons to the brain. It was also ascertained, in the case of delayed ingrowth, whether application of nerve growth factor immediately after lesion influenced the cell death process. This study confirmed that retinal ganglion cell death is a relatively late event in regeneration, because in several animals where anterograde HRP labeling demonstrated regenerating axons within the tectum, no cell death had occurred. There was no statistically significant difference in cell death at 75 days after lesion between animals receiving nerve crush and those receiving nerve cut with stump separation, even though most crush animals had regenerated a complete visual projection, whereas most nerve cut animals had not. The application of NGF did not influence the level of cell death at 75 days after lesion. These results suggest that contact of optic axons with the optic tract or tectum is not necessary for retinal ganglion cell death to occur. However, this does not necessarily mean that contact with the brain is not involved with cell death during regeneration following nerve crush because it is possible that the mechanisms of cell death are different when axons are prevented from regenerating. Further investigations are therefore required to establish the reasons for this cell death.     

Peripheral nerve grafts to the frog optic tectum: a morphological study of foreign axon regeneration in the central nervous system

The proximal stump of a transected mandibular nerve was grafted onto the rostrodorsal surface of the optic tectum in adult Rana pipiens to investigate the morphologic characteristics of nonspecific axonal regeneration in a highly organized region of central nervous system (CNS). Within the first 3 weeks postgraft surgery (WPS), the nerve-tectum interface became firmly established. Concomitant with this was an invasion of the host tectum by a small number of fine "pioneerlike" axons from the nerve. By 6 WPS there developed a concerted instreaming of a large number of peripheral fibers. Once within the CNS, the foreign axons distributed themselves throughout the rostrocaudal extent of the tectum, but primarily its dorsal aspect within superficial layers 8 and 9. Presence of intact optic fibers at the time of mandibular fiber invasion served somewhat to restrict the regenerating aberrant axons in their course through layer 9. This restriction could be avoided by removal of the optic input either before or during peripheral ingrowth. However, once peripheral fibers had entered and established themselves in the host environment, no subsequent manipulation of the retinotectal projection had any effect. The aberrant growth pattern, which appeared remarkably stable after 6 WPS, consisted of a plexus of medium- and fine-caliber peripheral axons. Many of these fibers had numerous branches and "en passant" varicosities, the latter encompassing a variety of shapes and sizes. Terminal swellings and arborizations were also found. When comparing the regeneration of optic and mandibular nerve fibers in the tectum, two distinctions were made. Whereas optic axons revealed a fascicular and layered organization, mandibular axons showed a highly segregated and disordered growth pattern. These characteristic differences were maintained even when the two fiber systems were allowed to coregenerate into the same target tectum. Thus, each of the two groups of axons interacts with the tectal substrate in a distinct manner, apparently independent of the other.     

A magnetic evaluation of peripheral nerve regeneration: II. The signal amplitude in the distal segment in relation to functional recovery

Motor and sensory function in a healthy nerve is strongly related to the number of neuronal units connecting to the distal target organs. In the regenerating nerve the amplitudes of magnetically recorded nerve compound action currents (NCACs) seem to relate to the number of functional neuronal units with larger diameters regenerating across the lesion. The goal of this experiment was to compare the signal amplitudes recorded from the distal segment of a reconstructed nerve to functional recovery. To this end, the peroneal nerves of 30 rabbits were unilaterally transected and reconstructed. After 6, 8, 12, 20, and 36 weeks of regeneration time the functional recovery was studied based on the toe-spread test, and the nerve regeneration based on the magnetically recorded NCACs. The results demonstrate that the signal amplitudes recorded magnetically from the reconstructed nerves increase in the first 12 weeks from 0% to 21% of the amplitudes recorded from the control nerves and from 21% to 25% in the following 23 weeks. The functional recovery increases from absent to good between the 8th and the 20th week after the reconstruction. A statistically significant relation was demonstrated between the signal amplitude and the functional recovery (P < 0.001). It is concluded that the magnetic recording technique can be used to evaluate the quality of a peripheral nerve reconstruction and seems to be able to predict, shortly after the reconstruction, the eventual functional recovery. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:750–755, 1998.     

Activation of fibers via experimentally produced stump neuromas of skin nerves: Ephaptic transmission or retrograde sprouting?

Fibers terminating in experimentally produced stump neuromas of the superficial peroneal nerve (SP, skin nerve) were investigated 6 to 245 days after cutting and ligating the nerve. The fibers were recorded from 60 to 80 mm cranial to the neuroma and tested to find whether or not they could be activated via the neuroma by electrical stimulation of the common peroneal nerve cranial to the recording site and from the superficial peroneal nerve distal to the recording site. Particularly unmyelinated and thin myelinated fibers could be activated via the neuromas by stimulation of unmyelinated and myelinated fibers. The discharges elicited via the neuroma from both stimulation sites had constant latencies and thresholds. They followed trains of high-frequency stimulation, depending on the conduction velocities of the recorded axons. Some axons could be excited at more than one latency and threshold whereby only one response could be elicited at a time. The frequency of this phenomenon was rare in the first 30 days after the nerve cut (about 2% of all thin axons) and attained values of 15 to 25% in the experiments carried out 105 to 245 days after the nerve cut. Sympathetic postganglionic fibers were not involved. The findings in this study can be explained by ephaptic transmission between axons in the neuromas or by retrograde sprouting of the fibers in the nerve for considerable distances or by both processes.     

Adenoviral vector-mediated expression of a foreign gene in peripheral nerve tissue bridges implanted in the injured peripheral and central nervous system

Axons of the CNS do normally not regenerate after injury, in contrast to axons of the PNS. This is due to a different microenvironment at the site of the lesion as well as a particular intrinsic program of axonal regrowth. Although transplantation of peripheral nerve tissue bridges is perhaps the most successful approach to promoting regeneration in the CNS, ingrowth of CNS nerve fibers with such transplants is limited. Genetic modification of peripheral nerve bridges to overexpress outgrowth-promoting proteins should, in principle, improve the permissive properties of peripheral nerve transplants. The present study shows that pieces of peripheral intercostal nerve, subjected to ex vivo adenoviral vector-mediated gene transfer and implanted as nerve bridges in transected sciatic nerve, avulsed ventral root, hemi-sected spinal cord and intact brain, are capable of expressing a foreign gene. In vitro studies showed expression of the reporter gene LacZ up to 30 days in Schwann cells. After implantation, LacZ expression could be detected at 7 days postimplantation, but had virtually disappeared at 14 days. Schwann cells of the transduced nerve bridges retained the capacity of guiding regenerative peripheral and central nerve fiber ingrowth. Transduction of intercostal nerve pieces prior to implantation should, in principle, enable enhanced local production of neurotrophic factors within the transplant and has the potential to improve the regeneration of injured axons into the graft.     

Long-term survival of glial segments during nerve regeneration in the leech

Nerve injury that severs axons also disrupts ensheathing glial cells. Specifically, crushing or cutting the leech nerve cord separates the glial cell's nucleated portion from an anucleate segment, or ‘stump'. In experiments reported here, the fate of the stump and the response of the nucleated portion were determined by intracellular recording, by intracellular injection of Lucifer Yellow dye and horseradish peroxidase (HRP) as tracers, and by electron microscopy. The nucleated portion of the glial cell did not divide, degenerate, or grow appreciably. The severed glial stump remained isolated from the nucleated portion but maintained its resting potential and normal morphology for months. Stumps typically began to deteriorate after 3 months. Small macrophage-like cells, or ‘microglia’ increased in number after injury and ensheathed axons, thus partially replacing the atrophying glial stump. Some axons in the nerve cord degenerated; the remainder appeared morphologically and physiologically normal. Thus, both nucleated and anucleate glial segments persisted throughout the one to two months required for axons to regenerate functional connections. Glial cells in the leech are therefore available to guide physically the growing axons or to contribute in other ways to nerve regeneration.     

Guinea pigs as an animal model for sciatic nerve injury

The overwhelming use of rat models in nerve regeneration studies is likely to induce skewness in treatment outcomes.To address the problem,this study was conducted in 8 adult guinea pigs of either sex to investigate the suitability of guinea pig as an alternative model for nerve regeneration studies.A crush injury was inflicted to the sciatic nerve of the left limb,which led to significant decrease in the pain perception and neurorecovery up to the 4th weak.Lengthening of foot print and shortening of toe spread were observed in the paw after nerve injury.A 3.49 ± 0.35 fold increase in expression of neuropilin 1(NRP1) gene and 2.09 ± 0.51 fold increase in neuropilin 2(NRP2) gene were recorded 1 week after nerve injury as compared to the normal nerve.Ratios of gastrocnemius muscle weight and volume of the experimental limb to control limb showed more than 50% decrease on the 30 th day.Histopathologically,vacuolated appearance of the nerve was observed with presence of degenerated myelin debris in digestion chambers.Gastrocnemius muscle also showed degenerative changes.Scanning electron microscopy revealed loose and rough arrangement of connective tissue fibrils and presence of large spherical globules in crushed sciatic nerve.The findings suggest that guinea pigs could be used as an alternative animal model for nerve regeneration studies and might be preferred over rats due to their cooperative nature while recording different parameters.     

Improved sciatic nerve regeneration by local thyroid hormone treatment in adult rat is accompanied by increased expression of SCG10

Thyroid hormone plays an important role in regulating the development and regeneration of the nervous system. Our previous work showed that local administration of triiodothyronine (T3) at the level of transected rat sciatic nerve increased the number and diameter of regenerated axons, but the mechanism underlying the improved regeneration is still unclear. Here, we have investigated the effect of T3 on the expression of SCG10, a regulator of microtubule dynamics in growth cones. After transection of adult rat sciatic nerves, silicone tubes were implanted and filled with T3 or phosphate-buffered solution. At various time points following surgery, the expression of SCG10 protein and mRNA was analyzed. Semi-quantitative Western blot analysis revealed that sciatic nerve transection induced a more than 20-fold upregulation of SCG10 protein in proximal nerve segments at 1 day post-lesion, while at this time point, SCG10 mRNA in dorsal root ganglion neurons was not increased yet. The increase in SCG10 protein and mRNA could be observed over 30 days. Local T3 treatment significantly enhanced the increase in SCG10 protein levels about two-fold in the different segments of transected nerve during the regeneration period. Also SCG10 mRNA levels in lumbar ganglia were enhanced. Immunohistochemical analysis showed that T3 treatment not only increased the number of SCG10 positive axons but also the intensity of their staining. These results suggest that SCG10 is involved in the regulation of regeneration. The stimulating effect of T3 on SCG10 expression could provide a mechanism by which T3 enhances peripheral nerve regeneration.     

A comparison of magnetic and electrical stimulation of peripheral nerves

We compared magnetic stimulation using different coil designs (2 rounded coils and a butterfly-prototype coil) with electrical stimulation of the median and ulnar nerves in 5 normal subjects. Using magnetic stimulation we were able to record technically satisfactory maximal sensory and motor responses only with the butterfly coil. Submaximal electrical stimuli preferentially activated sensory rather than motor axons, but submaximal magnetic stimuli did not. The onset latency, amplitude, area and duration of responses elicited electrically or magnetically with the butterfly coil during routine sensory and motor nerve conduction studies were similar, and motor and sensory conduction velocities were comparable when studied over long segments of nerve. However, the motor conduction velocities with magnetic and electrical stimulation differed by as much as 18 m/sec in the across-elbow segment of ulnar nerve. Thus, recent developments in magnetic stimulator design have improved the focality of the stimulus, but the present butterfly coil design cannot replace electrical stimulation for the detection of focal changes in nerve conduction velocity at common entrapment sites, such as in the across-elbow segment of the ulnar nerve.     

Recovery of neurophysiological features with time after rat sciatic nerve repair: a magneto-neurographic study

Experimental assessment of peripheral nerve regeneration in rats by electrophysiology is controversial due to low reproducibility of electrophysiological indicators and diminished quantitative evaluation in conventional experimental set-ups. Magnetoneurography (MNG) counteracts these drawbacks by magnetically recording electrophysiological signals ex vivo, thereby providing accurate and quantitative data. In 50 rats, sciatic nerve transection was followed by direct repair. MNG outcome parameters, footprints [static toe spread factor (TSF); function] and muscle weight (MW) were studied for their recovery pattern from 2 to 24 weeks. By using MNG, we showed that the regeneration process still continues when functional recovery (static TSF) becomes stagnant. With regression analysis, MNG parameters amplitude, amplitude area and conduction velocity (CV) demonstrated moderate significant correlation with MW, whereas CV was not significantly associated with static TSF. No significant association exists between MW and static TSF. A Kaplan-Meier survival curve revealed that autotomy/contracture of rat hind paws was not related to decreased MNG outcome values. In conclusion, this study highlights and discusses the dissimilarities between direct (MNG) and indirect (static TSF and MW) assessment techniques of the regeneration process. We emphasise the significance of MNG as a direct derivative of axon regeneration in experimental rat studies. Additionally, we stress the must for right-left ratios, as neurophysiological indicators vary with age, and we confute possible bias in footprint analysis caused by exclusion of autotomy/contracture animals.     

Fibrin glue repair leads to enhanced axonal elongation during early peripheral nerve regeneration in an in vivo mouse model

Microsurgical suturing is the gold standard of nerve coaptation. Although literature on the usefulness of ifbrin glue as an alternative is becoming increasingly available, it remains contradic-tory. Fu...     

Interleukin-10 reduces scarring and enhances regeneration at a site of sciatic nerve repair

Abstract   Axonal regeneration at a site of peripheral nerve repair can be impeded by the formation of scar tissue, which creates a mechanical barrier and initiates the development of multiple branched axonal sprouts that form a neuroma. We have investigated the hypothesis that the application of a scar-reducing agent to the nerve repair site would permit better axonal regeneration. In anaesthetised C57 Black-6 mice, the left sciatic nerve was sectioned and immediately re-approximated using four epineurial sutures. In five groups of eight mice, we injected transforming growth factor-β3 (50 or 500 ng), interleukin-10 (IL-10) (125 or 500 ng), or saline into and around the repair site, both before and after the nerve section. Another group of eight animals acted as sham-operated controls. After 6 weeks, the outcome was assessed by recording compound action potentials (CAPs), measuring collagen levels using picrosirius red staining, and counting the number of myelinated axons proximal and distal to the repair. CAPs evoked by electrical stimulation distal to the repair were significantly smaller in all repair groups except for the low-dose IL-10 group, where they were not significantly different from that in controls. The area of staining for collagen had significantly increased in all repair groups except for the low-dose IL-10 group, which was not significantly different from that in controls. The myelinated fibre counts were always higher distal to the repair site, but there were no significant differences between groups. We conclude that administration of a low-dose of IL-10 to a site of sciatic nerve repair reduces scar formation and permits better regeneration of the damaged axons.     

Studies of the early stages of optic axon regeneration in the goldfish

We have studied the early stages (4-14 days) of axonal regeneration following intraorbital optic nerve crush in the goldfish. We used 3H-proline autoradiography to anterogradely label and visualize the growing axons and wheat germ agglutinin-conjugated horseradish peroxidase (WGA:HRP) for retrograde labeling to determine the cells of origin of the earliest projections. The first retinal ganglion cells (RGCs) that could be retrogradely filled from the optic tract, following optic nerve crush, were in the central retina and were seen at 8 days postoperative. More peripheral cells were only labeled with longer postcrush survival periods. Thus, the first axons to regenerate past the lesion were from central RGCs. The axons of these cells extended into the cranial nerve stump between 4 and 5 days postcrush and entered the nerve as a fascicle, which travelled just beneath its surface. Studies of nerve cross sections from animals at 5-8 days postoperative demonstrated that initial outgrowth was not confined to any particular locale within the nerve although the early fibers appeared to avoid its temporal aspect. When the regenerating axons reached the optic tract they remained in fascicles but left the surface to run along the medial, deep portion of the tract, immediately adjacent to the diencephalon and pretectum. The positions occupied by the earliest-regenerating axons in the optic nerve were variable and not always appropriate for their central retinal origin. However, the abrupt change in growth trajectory as the fibers entered the optic tract brought them into the areas of the visual paths that are occupied by central axons in intact animals. We suggest that this change in position is related to both changes in the structural organization of the intracranial visual paths and to possible axon guidance signals in the region of the nerve-tract juncture.     

Polymer electret guidance channels enhance peripheral nerve regeneration in mice

Polytetrafluoroethylene (PTFE) tubes were prepared as electrets displaying a quasi-permanent surface charge due to the presence of trapped monopolar charge carriers. PTFE tubes containing either positive or negative charges and electrically neutral PTFE tubes were used as nerve guidance channels for the repair of a ⁴mm nerve gap in the sciatic nerve of mice. After 4 weeks of implantation, positively and negatively charged PTFE electrets contained regenerated nerves with significantly more myelinated axons than nerves regenerated in uncharged PTFE tubes. This observation suggests that peripheral nerve regeneration can be enhanced by electrically charged nerve guidance channels.