Studying nerve transfers: Searching for a consensus in nerve axons count.

Axonal count is the base for efficient nerve transfer; despite its capital importance, few studies have been published on human material, most research approaches being performed on experimental animal models of nerve injury. Thus, standard analysis methods are still lacking. Quantitative data obtained have to be reproducible and comparable with published data by other research groups. To share results with the scientific community, the standardization of quantitative analysis is a fundamental step. For this purpose, the experiences of the Italian, Austrian, German, Greek, and Iberian-Latin American groups have been compared with each other and with the existing literature to reach a consensus in the fiber count and draw up a protocol that can make future studies from different centers comparable. The search for a standardization of the methodology was aimed to reduce all the factors that are associated with an increase in the variability of the results. All the preferential methods to be used have been suggested. On the other hand, alternative methods and different methods have been identified to achieve the same goal, which in our experience are completely comparable; therefore, they can be used indifferently by the different centers according to their experience and availability.

Cadaveric Evaluation of Myelinated Nerve Fiber Count in the Nerve to the Gracilis Muscle in Relation to Use as a Free Functional Muscle Transfer for Elbow Flexion.

BACKGROUND: Optimizing axon count is essential for successful nerve transfer surgery, and a donor-to-recipient axon count ratio greater than 0.7:1 has been associated with improved outcomes. A gracilis free functioning muscle transfer (FFMT) is an option to restore elbow flexion, but its axon count has not been evaluated. Our aim was to quantify the axon count of the nerve to the gracilis muscle. METHODS: The nerve to the gracilis was dissected in 10 fresh frozen adult cadaveric hindquarter specimens (four females and six males). The length of the nerve to the gracilis was measured and a biopsy taken. A validated histologic preparation technique was utilized, and axons were counted. The mean length and axon counts were calculated. RESULTS: The average axon count in the nerve to the gracilis was 818 (range = 684-1,000, standard deviation [SD] = 116). The average length was 98 mm (range = 81-115 mm, SD = 13 mm). CONCLUSION: Our study found the average axon count in the nerve to the gracilis was 818. Prior literature suggests axon count ratio greater than 0.7:1 is associated with better clinical outcomes. Using data from prior studies, the spinal accessory, three intercostal, and two intercostal nerves are all sufficient for the transfer to the nerve to the gracilis with donor to recipient ratios of 1.7:1, 1.3:1, and 0.9:1, respectively.

Significance of the Marginal Mandibular Branch in Relation to Facial Palsy Reconstruction: Assessment of Microanatomy and Macroanatomy Including Axonal Load in 96 Facial Halves.

BACKGROUND: The marginal mandibular branch (MMB) of the facial nerve provides lower lip symmetry apparent during human smile or crying and is mandatory for vocal phonation. In treating facial palsy patients, so far, little attention is directed at the MMB in facial reanimation surgery. However, isolated paralysis may occur congenital, in Bell's palsy or iatrogenic during surgery, prone to its anatomical course. A variety of therapies address symmetry with either weakening of the functional side or reconstruction of the paralyzed side. To further clarify the histoanatomic basis of facial reanimation procedures using nerve transfers, we conducted a human cadaver study examining macroanatomical and microanatomical features of the MMB including its axonal capacity. METHODS: Nerve biopsies of the MMB were available from 96 facial halves. Histological processing, digitalization, nerve morphometry investigation, and semiautomated axonal quantification were performed. Statistical analysis was conducted with P < 0.05 as level of significance. RESULTS: The main branch of 96 specimens contained an average of 3.72 fascicles 1 to 12, and the axonal capacity was 1603 ± 849 (398-5110, n = 85). Differences were found for sex (P = 0.018), not for facial sides (P = 0.687). Diameters were measured with 1130 ± 327 µm (643-2139, n = 79). A significant difference was noted between sexes (P = 0.029), not for facial sides (P = 0.512.) One millimeter in diameter corresponded to 1480 ± 630 axons (n = 71). A number of 900 axons was correlated with 0.97 mm (specificity, 90%; sensitivity, 72%). CONCLUSIONS: Our morphometric results for the MMB provide basic information for further investigations, among dealing with functional reconstructive procedures such as nerve transfers, nerve grafting for direct neurotization or babysitter procedures, and neurectomies to provide ideal power and authenticity.

A Rapid Protocol for Intraoperative Assessment of Peripheral Nerve Myelinated Axon Count and Its Application to Cross-Facial Nerve Grafting.

BACKGROUND: Donor nerve myelinated axon counts correlate with functional outcomes in reanimation procedures; however, there exists no reliable means for their intraoperative quantification. In this article, the authors report a novel protocol for rapid quantification of myelinated axons from frozen sections, and demonstrate its applicability to surgical practice. METHODS: The impact of various fixation and FluoroMyelin Red staining strategies on resolved myelin sheath morphology from cryosections of rat and rabbit femoral and sciatic nerves was assessed. A protocol comprising fresh cryosection and rapid staining was developed, and histomorphometric results were compared against conventional osmium-postfixed, resin-embedded, toluidine blue-stained sections of rat sciatic nerve. The rapid protocol was applied for intraoperative quantification of donor nerve myelinated axon count in a cross-facial nerve grafting procedure. RESULTS: Resolution of myelinated axon morphology suitable for counting was realized within 10 minutes of tissue harvest. Although mean myelinated axon diameter appeared larger using the rapid fresh-frozen as compared to conventional nerve processing techniques (mean ± SD; rapid, 9.25 ± 0.62 µm; conventional, 6.05 ± 0.71 µm; p < 0.001), no difference in axon counts was observed on high-power fields (rapid, 429.42 ± 49.32; conventional, 460.32 ± 69.96; p = 0.277). Whole nerve myelinated axon counts using the rapid protocol herein (8435.12 ± 1329.72) were similar to prior reports using conventional osmium processing of rat sciatic nerve. CONCLUSIONS: A rapid protocol for quantification of myelinated axon counts from peripheral nerves using widely available equipment and techniques has been described, rendering possible intraoperative assessment of donor nerve suitability for reanimation.

Anatomic characteristics of supraorbital and supratrochlear nerves relevant to their use in corneal neurotization.

BACKGROUND: Corneal denervation can lead to opacification and blindness. A new treatment technique, surgical corneal neurotization, transfers healthy donor nerve, (most commonly contralateral supratrochlear or supraorbital) to the affected limbus to prevent corneal destruction and improve healing potential of the cornea following insult. We examine gross and histomorphometric anatomy of the supratrochlear and supraorbital nerves relevant to their use in corneal neurotization. METHODS: For each of nine adult cadaver heads, bilateral supraorbital and supratrochlear nerves were dissected from the supraorbital rim to the anterior hairline. The following data were recorded for each nerve: exit from the orbit through a notch versus foramen; horizontal distance from midline at the supraorbital rim; and distance from orbital exit to first branching point. Samples of all left supraorbital and supratrochlear nerves were obtained at the level of the supraorbital rim and at points 3 cm and 6 cm distally for histomorphometric analysis. Myelinated axon counts were determined for each sample. RESULTS: Four supraorbital foramina, 14 supraorbital notches, two supratrochlear foramina, and 15 supratrochlear notches were identified. Average supraorbital and supratrochlear distances to midline were 26.5 mm and 21 mm respectively. Average myelinated axon counts for both nerves were greater at the orbital rim (supraorbital: 6018, supratrochlear: 2533) than at 6 cm distally (supraorbital: 1621, supratrochlear: 1112). CONCLUSIONS: Anatomic dissection shows relative close approximation of the supraorbital and supratrochlear nerves, with a high proportion of both nerves exiting the orbit through foramina. The supraorbital nerve at the orbital rim contains the greatest number of myelinated axons.

Axon numbers and landmarks of trigeminal donor nerves for corneal neurotization.

PURPOSE: Corneal anesthesia leads to chronic corneal injury. This anatomical study characterizes the donor nerve branches of the supratrochlear and supraorbital nerves used for corneal neurotization. METHODS: In 13 non-embalmed cadavers, the supratrochlear and supraorbital nerves were dissected and distances to anatomical landmarks measured. Cross-sections of supratrochlear and supraorbital donor nerves were harvested and histomorphometrically analyzed to assess the number of myelinated axons. RESULTS: The donor axon counts were 3146 ± 1069.9 for the supratrochlear and 1882 ± 903 for the supraorbital nerve distal to the supraorbital notch. The supratrochlear nerve was dissected on the medial upper eyelid 2 cm lateral to the facial midline and the branch of the supraorbital nerve 1 cm medial to the mid-pupillary line. CONCLUSION: The supraorbital and supratrochlear branches of the trigeminal nerve are potent donor nerves for corneal neurotization in the treatment of neuropathic keratopathy and can be reliably dissected using anatomical landmarks.

Side-To-Side Nerve Bridges Support Donor Axon Regeneration Into Chronically Denervated Nerves and Are Associated With Characteristic Changes in Schwann Cell Phenotype.

BACKGROUND: Chronic denervation resulting from long nerve regeneration times and distances contributes greatly to suboptimal outcomes following nerve injuries. Recent studies showed that multiple nerve grafts inserted between an intact donor nerve and a denervated distal recipient nerve stump (termed "side-to-side nerve bridges") enhanced regeneration after delayed nerve repair. OBJECTIVE: To examine the cellular aspects of axon growth across these bridges to explore the "protective" mechanism of donor axons on chronically denervated Schwann cells. METHODS: In Sprague Dawley rats, 3 side-to-side nerve bridges were placed over a 10-mm distance between an intact donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) distal nerve stump. Green fluorescent protein-expressing TIB axons grew across the bridges and were counted in cross section after 4 weeks. Immunofluorescent axons and Schwann cells were imaged over a 4-month period. RESULTS: Denervated Schwann cells dedifferentiated to a proliferative, nonmyelinating phenotype within the bridges and the recipient denervated CP nerve stump. As donor TIB axons grew across the 3 side-to-side nerve bridges and into the denervated CP nerve, the Schwann cells redifferentiated to the myelinating phenotype. Bridge placement led to an increased mass of hind limb anterior compartment muscles after 4 months of denervation compared with muscles whose CP nerve was not "protected" by bridges. CONCLUSION: This study describes patterns of donor axon regeneration and myelination in the denervated recipient nerve stump and supports a mechanism where these donor axons sustain a proregenerative state to prevent deterioration in the face of chronic denervation.

Spider silk as guiding biomaterial for human model neurons.

Over the last years, a number of therapeutic strategies have emerged to promote axonal regeneration. An attractive strategy is the implantation of biodegradable and nonimmunogenic artificial scaffolds into injured peripheral nerves. In previous studies, transplantation of decellularized veins filled with spider silk for bridging critical size nerve defects resulted in axonal regeneration and remyelination by invading endogenous Schwann cells. Detailed interaction of elongating neurons and the spider silk as guidance material is unknown. To visualize direct cellular interactions between spider silk and neurons in vitro, we developed an in vitro crossed silk fiber array. Here, we describe in detail for the first time that human (NT2) model neurons attach to silk scaffolds. Extending neurites can bridge gaps between single silk fibers and elongate afterwards on the neighboring fiber. Culturing human neurons on the silk arrays led to an increasing migration and adhesion of neuronal cell bodies to the spider silk fibers. Within three to four weeks, clustered somata and extending neurites formed ganglion-like cell structures. Microscopic imaging of human neurons on the crossed fiber arrays in vitro will allow for a more efficient development of methods to maximize cell adhesion and neurite growth on spider silk prior to transplantation studies.

The therapeutic effects of human adipose-derived stem cells in a rat cervical spinal cord injury model.

Spinal cord injury triggers a cascade of degenerative changes leading to cell death and cavitation. Severed axons fail to regenerate across the scar tissue and are only capable of limited sprouting. In this study, we investigated the effects of adult human adipose-derived stem cells (ASC) on axonal regeneration following transplantation into the injured rat cervical spinal cord. ASC did not induce activation of astrocytes in culture and supported neurite outgrowth from adult rat sensory dorsal root ganglia neurons. After transplantation into the lateral funiculus 1 mm rostral and caudal to the cervical C3-C4 hemisection, ASC continued to express brain-derived neurotrophic factor, vascular endothelial growth factor, and fibroblast growth factor-2 for 3 weeks but only in animals treated with cyclosporine A. Transplanted ASC stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone with some terminal arborizations reaching the caudal spinal cord. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure of the lesion scar with numerous astrocytic processes extended into the middle of the trauma zone in a chain-like pattern and in close association with regenerating axons. The density of the astrocytic network was also significantly decreased. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the activity of OX42-positive microglial cells was markedly reduced. However, ASC did not support recovery of forelimb function. The results suggest that transplanted ASC can modify the structure of the glial scar and stimulate axonal sprouting.

Combined treatment with platelet-rich plasma and brain-derived neurotrophic factor-overexpressing bone marrow stromal cells supports axonal remyelination in a rat spinal cord hemi-section model.

BACKGROUND AIMS: Combining biologic matrices is becoming a better choice to advance stem cell-based therapies. Platelet-rich plasma (PRP) is a biologic product of concentrated platelets and has been used to promote regeneration of peripheral nerves after injury. We examined whether PRP could induce rat bone marrow stromal cells (BMSCs) differentiation in vitro and whether a combination of BMSCs, PRP and brain-derived neurotrophic factor (BDNF) could provide additive therapeutic benefits in vivo after spinal cord injury (SCI). METHODS: BMSCs and BDNF-secreting BMSCs (BDNF-BMSCs) were cultured with PRP for 7 days and 21 days, respectively, and neurofilament (NF)-200, glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2) and ribosomal protein S6 kinase (p70S6K) gene levels were assessed. After T10 hemi-section in 102 rats, 15-µL scaffolds (PRP alone, BMSCs, PRP/BMSCs, BDNF-BMSCs or PRP/BDNF-BMSCs) were transplanted into the lesion area, and real-time polymerase chain reaction, Western blot, immunohistochemistry and ultrastructural studies were performed. RESULTS: The messenger RNA expression of NF-200, GFAP, MAP2 and p70S6K was promoted in BMSCs and BDNF-BMSCs after culture with PRP in vitro. BDNF levels were significantly higher in the injured spinal cord after implantation of BDNF-BMSCs. In the PRP/BDNF-BMSCs group at 8 weeks postoperatively, more GFAP was observed, with less accumulation of astrocytes at the graft-host interface. Rats that received PRP and BDNF-BMSC implants showed enhanced hind limb locomotor performance at 8 weeks postoperatively compared with control animals, with more axonal remyelination. CONCLUSIONS: A combined treatment comprising PRP and BDNF-overexpressing BMSCs produced beneficial effects in rats with regard to functional recovery after SCI through enhancing migration of astrocytes into the transplants and axonal remyelination.

Regeneration in, and properties of, extracted peripheral nerve allografts and xenografts.

When not enough conventional autologous nerve grafts are available, alternatives are needed to bridge nerve defects. Our aim was to study regeneration of nerves in chemically-extracted acellular nerve grafts from frogs, mice, humans (fresh and stored sural nerve), pigs and rats when defects in rat sciatic nerves were bridged. Secondly, we compared two different extraction procedures (techniques described by Sondell et al. and Hudson et al.) with respect to how efficiently they supported axonal outgrowth, and remaining laminin and myelin basic protein (MBP), after extraction. Isografts (rat) and xenografts (mouse) were transplanted into defects in rat sciatic nerves. Acellular nerve allografts from rats, extracted by the Sondell et al's technique, had an appreciably longer axonal outgrowth based on immunohistochemical staining of neurofilaments, than acellular nerve xenografts except those from the pig. Among acellular xenografts there was considerably longer axonal outgrowth in the grafts from pigs compared with those from humans (fresh), but there were no other differences among the xenografts with respect to axonal outgrowth. Axonal outgrowth in acellular nerve xenografts from mice, extracted by the method described by Sondell et al. was longer than in those extracted by Hudson et al's method, while there was no difference in outgrowth between extracted nerve isografts from rats. Electrophoretic analysis of extracted acellular nerve grafts showed remaining laminin, but not MBP, after both extraction procedures. These preserved laminin and removed MBP in acellular nerve grafts. Such grafts can be used to reconstruct short defects in nerves irrespective of their origin. However, selecting and matching a suitable combination of graft and host species may improve axonal outgrowth.

Olfactory ensheathing cells can reduce the tissue loss but not the cavity formation in contused spinal cord of rats.

In recent years, olfactory ensheathing cells (OECs) have been used as a therapeutic strategy to repair the anatomical structure and promote the function recovery of injured spinal cord in both animal and human. In this study, OECs were transplanted into contused spinal cords of adult rats. After dorsal laminectomy at T10 vertebra, spinal cord was injured by a force of 10 g with NYU II impactor from 25 mm above the exposed cord. The contused spinal cord received injections of OECs in DMEM or DMEM alone at one week after injury. The migration and distribution of OECs in the contused spinal cord were observed by the light microscope. The intact tissue area, injured tissue area, cavity size, number of myelinated nerve fibers and neurons labeled by CB-HRP in T8 segment were measured and counted by the semi-quantitative techniques at 6 weeks after transplantation. Locomotor ability and conductive function of the spinal cord were evaluated by the BBB score and cortical somatosensory evoked potentials (CSEP) recording. OECs were found in both lesion site and tissue near the lesion. The intact tissue area was significantly larger in the OECs-transplanted rats than that in the DMEM-injected animals, whereas the injured tissue area was significantly smaller in the OECs-rats than that in the DMEM-rats. The number of myelinated nerve fibers in the lesion site and preserved neurons in T8 was significantly greater in the OECs-group than in the DMEM-group, but the cavity size detected was not significantly different between the two groups. The BBB score and CSEP recording showed a better performance of locomotor ability and conductive function in the OECs-transplanted rats than in the DMEM-injected animals. These results indicate that OECs can counteract secondary tissue degeneration after spinal cord injury. Although they cannot reduce the cavity formation, they can promote morphological preservation and functional improvement of the contused spinal cord.

Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury.

Various types of induced pluripotent stem (iPS) cells have been established by different methods, and each type exhibits different biological properties. Before iPS cell-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Here we show the directed neural differentiation of murine iPS cells and examine their therapeutic potential in a mouse spinal cord injury (SCI) model. "Safe" iPS-derived neurospheres, which had been pre-evaluated as nontumorigenic by their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse brain, produced electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro. Furthermore, when the safe iPS-derived neurospheres were transplanted into the spinal cord 9 d after contusive injury, they differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in remyelination and induced the axonal regrowth of host 5HT(+) serotonergic fibers, promoting locomotor function recovery. However, the transplantation of iPS-derived neurospheres pre-evaluated as "unsafe" showed robust teratoma formation and sudden locomotor functional loss after functional recovery in the SCI model. These findings suggest that pre-evaluated safe iPS clone-derived neural stem/progenitor cells may be a promising cell source for transplantation therapy for SCI.

Peripheral nerve grafts after cervical spinal cord injury in adult cats.

Peripheral nerve grafts (PNG) into the rat spinal cord support axon regeneration after acute or chronic injury, with synaptic reconnection across the lesion site and some level of behavioral recovery. Here, we grafted a peripheral nerve into the injured spinal cord of cats as a preclinical treatment approach to promote regeneration for eventual translational use. Adult female cats received a partial hemisection lesion at the cervical level (C7) and immediate apposition of an autologous tibial nerve segment to the lesion site. Five weeks later, a dorsal quadrant lesion was performed caudally (T1), the lesion site treated with chondroitinase ABC 2 days later to digest growth inhibiting extracellular matrix molecules, and the distal end of the PNG apposed to the injury site. After 4-20 weeks, the grafts survived in 10/12 animals with several thousand myelinated axons present in each graft. The distal end of 9/10 grafts was well apposed to the spinal cord and numerous axons extended beyond the lesion site. Intraspinal stimulation evoked compound action potentials in the graft with an appropriate latency illustrating normal axonal conduction of the regenerated axons. Although stimulation of the PNG failed to elicit responses in the spinal cord distal to the lesion site, the presence of c-Fos immunoreactive neurons close to the distal apposition site indicates that regenerated axons formed functional synapses with host neurons. This study demonstrates the successful application of a nerve grafting approach to promote regeneration after spinal cord injury in a non-rodent, large animal model.

Regenerated host axons form synapses with neurons derived from neural stem cells transplanted into peripheral nerves.

It is reported that neural stem cells (NSC) can arrest denervated muscle atrophy and promote nerve regeneration when transplanted into injured peripheral nerves, and that regenerated host axons can form synapses with transplanted and differentiated NSC. In this study, F344 rat nerve segments and F344 rat NSC were transplanted into host green fluorescence protein (GFP) transgenic F344 rats. This allowed transplanted F344 rat tissue to be used as a nonluminous background for the clear visualization of regenerated host GFP axons. Regenerated host axons grew into the transplanted F344 nerve segment 2 weeks after nerve anastomosis. Immunohistochemical staining and confocal microscope analysis revealed that regenerated host axons formed synapses with NSC-derived neurons. The findings confirmed that regenerated peripheral axons form synapses with neurons in peripheral nerves, possibly forming the basis for clinical application in peripheral nerve injury.

Transplanted embryonic spinal tissue promotes severed sciatic nerve regeneration in rats.

The effects of transplanted embryonic spinal tissue on host motor nerve regeneration and target muscle reinervation were investigated in severed sciatic nerves of rats. The electromyogram (EMG) responses and number of motor end plates (MEP) in target muscles, number of nerve axons, and retrogradely labeled motor neurons were examined in transplantation-, anastomosis without transplantation-, and naïve groups of the animals. The EMG patterns of the transplantation group returned to nearly normal at the 8th week, but those of the anastomosis group did not. MEP counts in the transplantation group were significantly higher than in the anastomosis group. The myelinated axon counts and myelin sheath thickness in the transplantation group were significantly higher than those in the anastomosis group. The number of retrogradely labeled motor neurons was significantly higher in the transplantation group. We conclude that transplanted embryonic spinal tissue can promote both host motor nerve regeneration and target muscle reinnervation.

Cytokine-induced SOCS expression is inhibited by cAMP analogue: impact on regeneration in injured retina.

Injured adult retinal ganglion cells (RGCs) regrow axons into peripheral nerve (PN) grafted onto cut optic nerve. Survival and regeneration of RGCs is increased by intraocular injections of ciliary neurotrophic factor (CNTF) and axonal regeneration is further enhanced by co-injection of a cyclic AMP analogue (CPT-cAMP). Based on these data, and because cytokine signaling is negatively regulated by suppressor of cytokine signaling (SOCS) proteins, we set out to determine whether CNTF injections increase retinal SOCS expression and whether any changes are attenuated by co-injection with CPT-cAMP. Using quantitative PCR we found increased SOCS1, SOCS2 and SOCS3 mRNA levels at various times after a single CNTF injection. Expression remained high for many days. SOCS protein levels were also increased. In situ hybridization revealed that RGCs express SOCS3 mRNA, and SOCS expression in cultured RGCs was increased by CNTF. Co-injection of CPT-cAMP reduced CNTF induced expression of SOCS1 and SOCS3 mRNA and decreased SOCS3 protein expression. CNTF injection also transiently increased retinal leukemia inhibitory factor (LIF) expression, an effect that was also moderated by CPT-cAMP. We propose that, along with known reparative effects of elevated cAMP on neurons, reducing SOCS upregulation may be an additional way in which cyclic nucleotides augment cytokine-induced regenerative responses in the injured CNS.

Accuracy of motor axon regeneration across autograft, single-lumen, and multichannel poly(lactic-co-glycolic acid) nerve tubes.

OBJECTIVE: The accuracy of motor axon regeneration becomes an important issue in the development of a nerve tube for motor nerve repair. Dispersion of regeneration across the nerve tube may lead to misdirection and polyinnervation. In this study, we present a series of methods to investigate the accuracy of regeneration, which we used to compare regeneration across autografts and single-lumen poly(lactic-co-glycolic acid) (PLGA) nerve tubes. We also present the concept of the multichannel nerve tube that may limit dispersion by separately guiding groups of regenerating axons. METHODS: The simultaneous tracing of the tibial and peroneal nerves with fast blue and diamidino yellow was performed 8 weeks after the repair of a 1-cm nerve gap in the rat sciatic nerve to determine the percentage of double-projecting motoneurons. Sequential tracing of the peroneal nerve with diamidino yellow 1 week before repair and fast blue 8 weeks after repair was performed to determine the percentage of correctly directed peroneal motoneurons. RESULTS: In the cases in which there was successful regeneration across single-lumen nerve tubes, more motoneurons had double projections to both the tibial and peroneal nerve branches after single-lumen nerve tube repair (21.4%) than after autograft repair (5.9%). After multichannel nerve tube repair, this percentage was slightly reduced (16.9%), although not significantly. The direction of regeneration was nonspecific after all types of repair. CONCLUSION: Retrograde tracing techniques provide new insights into the process of regeneration across nerve tubes. The methods and data presented in this study can be used as a basis for the development of a nerve tube for motor nerve repair.

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.

Experimental neurotisation of the brachial plexus by means of nerve grafting from the corticospinal tract of the proximal spinal cord at the T3-T4 level.

This paper reports experimental work in rats in which the radial nerve was cut at its origin from the brachial plexus and connected by means of a nerve graft to the corticospinal tract of the spinal cord at the T3-T4 level. After surgery, the triceps and extensor muscles of the forearm were able to extend the elbow and the wrist and gave almost normal responses on EMG. This reinnervation was no longer cholinergic, as demonstrated by the Vecuronium test. Because of the drawbacks associated with implantation into the spinal cord, this technique could not be used in all cases of total avulsion of the brachial plexus but it might be useful in those rare cases in which avulsion of all the cervical nerves of the brachial plexus is associated with paraplegia: such cases are rare but are occasionally seen.