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.

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.

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.

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.

Upregulation of activating transcription factor 3 (ATF3) by intrinsic CNS neurons regenerating axons into peripheral nerve grafts.

The expression of the transcription factor ATF3 in the brain was examined by immunohistochemistry during axonal regeneration induced by the implantation of pieces of peripheral nerve into the thalamus of adult rats. After 3 days, ATF3 immunoreactivity was present in many cells within approximately 500 mum of the graft. In addition, ATF3-positive cell nuclei were found in the thalamic reticular nucleus (TRN) and medial geniculate nuclear complex (MGN), from which most regenerating axons originate. CNS cells with ATF3-positive nuclei were predominantly neurons and did not show signs of apoptosis. The number of ATF3-positive cells had declined by 7 days and further by 1 month after grafting when most ATF3-positive cells were found in the TRN and MGN. 14 days or more after grafting, some ATF3-positive nuclei were distorted and may have been apoptotic. In some experiments of 1 month duration, neurons which had regenerated axons to the distal ends of grafts were retrogradely labeled with DiAsp. ATF3-positive neurons in these animals were located in regions of the TRN and MGN containing retrogradely labeled neurons and the great majority were also labeled with DiAsp. SCG10 and c-Jun were found in neurons in the same regions as retrogradely labeled and ATF3-positive cells. Thus, ATF3 is transiently upregulated by injured CNS neurons, but prolonged expression is part of the pattern of gene expression associated with axonal regeneration. The co-expression of ATF3 with c-jun suggests that interactions between these transcription factors may be important for controlling the program of gene expression necessary for regeneration.

Transplantation of bone marrow stromal cell-derived Schwann cells promotes axonal regeneration and functional recovery after complete transection of adult rat spinal cord.

The aim of this study was to evaluate whether transplantation of Schwann cells derived from bone marrow stromal cells (BMSC-SCs) promotes axonal regeneration and functional recovery in completely transected spinal cord in adult rats. Bone marrow stromal cells (BMSCs) were induced to differentiate into Schwann cells in vitro. A 4-mm segment of rat spinal cord was removed completely at the T7 level. An ultra-filtration membrane tube, filled with a mixture of Matrigel (MG) and BMSC-SCs (BMSC-SC group) or Matrigel alone (MG group), was grafted into the gap. In the BMSC-SC group, the number of neurofilament- and tyrosine hydroxylase-immunoreactive nerve fibers was significantly higher compared to the MG group, although 5-hydroxytryptamine- or calcitonin gene-related peptide-immunoreactive fibers were rarely detectable in both groups. In the BMSC-SC group, significant recovery of the hindlimb function was recognized, which was abolished by retransection of the graft 6 weeks after transplantation. These results demonstrate that transplantation of BMSC-SCs promotes axonal regeneration of lesioned spinal cord, resulting in recovery of hindlimb function in rats. Transplantation of BMSC-SCs is a potentially useful treatment for spinal cord injury.

Effect of number of fascicle on axonal regeneration in cable grafts.

The effect of number of fascicles on axonal regeneration in cable grafts was examined in the rat cable graft model. The study comprised three experimental groups: the 5f-group, which received 5 fascicles, larger than the host; the 3f-group, in which the total area of the graft fascicles was similar to that of the host; and the 1f-group, which received one fascicle cable graft, smaller in diameter than the host nerve. At the graft segment, well-myelinated fibers were observed both inside and outside the graft fascicles. The three groups showed no difference in morphometric and functional assessment, suggesting that the fibers which regenerated through the outside of the graft might be effectively induced into the distal host. The disproportionate enlargement of the graft fascicle of the 1f-group also increased the fibers passing through it. These findings suggest that a small number of fascicles can induce a larger population of regenerated fibers in the 20-mm cable graft model.

Peripheral olfactory ensheathing cells reduce scar and cavity formation and promote regeneration after spinal cord injury.

Bridging of a lesion site and minimizing local damage to create an environment permissive for regeneration are both primary components of a successful strategy to repair spinal cord injury (SCI). Olfactory ensheathing cells (OECs) are prime candidates for autologous transplantation to bridge this gap, but little is known currently about their mechanism of action. In addition, OECs from the accessible lamina propria (LP) of the olfactory mucosa are a more viable source in humans but have yet to be tested for their ability to promote regeneration in established SCI models. Here, mouse LP-OECs expressing green fluorescent protein (GFP) transplanted directly into both rat and mouse dorsolateral spinal cord lesion sites demonstrate limited migration but interact with host astrocytes to develop a new transitional zone at the lesion border. LP-OECs also promote extensive migration of host Schwann cells into the central nervous system repair zone and stimulate angiogenesis to provide a biological scaffold for repair. This novel environment created by transplanted and host glia within the spinal cord inhibits cavity and scar formation and promotes extensive sprouting of multiple sensory and motor axons into and through the lesion site. Sixty days after rat SCI, serotonin- and tyrosine hydroxylase-positive axons sprouted across the lesion into the distal cord, although axotomized rubrospinal axons did not. Thus, even in a xenotransplant paradigm, LP-OECs work collaboratively with host glial cells to create an environment to ameliorate local damage and simultaneously promote a regenerative response in multiple axonal populations.

Grafted swine neuroepithelial stem cells can form myelinated axons and both efferent and afferent synapses with xenogeneic rat neurons.

Neuroepithelial stem cells derived from the swine mesencephalic neural tube were examined regarding their eligibility for neural xenografting as a donor material, with the aim of evaluating myelinated axon formation and both types of synaptic formation with xenogeneic host neurons as part of possible neural circuit reconstruction. The mesencephalic neural tube tissues were dissected out from swine embryos at embryonic days 17 and 18 and were implanted immediately into the striatum of the Parkinsonian model rat. The swine-derived grafts had many nestin-positive rosette-forming, neurofilament-positive, and tyrosine hydroxylase-positive cells in the rat striatum. Electron microscopic study revealed both efferent and afferent synaptic formations in the donor-derived immature neurons or tyrosine hydroxylase-positive donor cells in the grafts. Myelinated axons, both positive and negative for swine-specific neurofilament antibody, were mingled together in the graft. These results indicated that implanted neuroepithelial stem cells could survive well and divide asymmetrically into both nestin-expressing precursors and differentiated neurochemical marker-expressing neurons in the xenogeneic rat striatum, with the help of an immunosuppressant. Donor-derived immature neurons formed both efferent and afferent synapses with xenogeneic host neurons, and donor-derived axons were myelinated, which suggests that implanted swine neuroepithelial stem cells could possibly restore damaged neuronal circuitry in the diseased brain.

Functional impact of axonal misdirection after peripheral nerve injuries followed by graft or tube repair.

Accuracy of reinnervation is one of the main factors conditioning functional recovery after brain, spinal, or peripheral axonal damage. Using the peripheral nerve as an experimental model, we studied the amount of inaccurate muscle reinnervation and its consequences on walking. Adult rats were submitted to an 8-mm resection of the sciatic nerve repaired by autograft (AG, n = 9), silicone (SIL, n = 13) or poly-L-lactate-epsilon-caprolactone (PLC, n = 11) single guides, and fascicular tubulization of peroneal and tibial branches with a dual silicone tube (FSIL, n = 9). At the end of follow-up, the sciatic nerve and its tibial and peroneal fascicles were dissected and stimulated by means of a suction electrode. In control rats, gastrocnemius and plantar muscles are fully innervated by the tibial fascicle and the tibialis anterior muscle by the peroneal nerve. None of the groups had noticeable recovery of locomotion assessed by the walking track index (SFI around -70 in all groups). After resection, all animals of groups AG, SIL, and PLC showed aberrant muscle reinnervation by axons from a non-corresponding fascicle, whereas in group FSIL only one of six regenerated animals showed misdirected activity. The proportion of inaccurate muscle activation was similar in group AG (47% for gastrocnemius, 54% for tibialis anterior, and 44% for plantar muscles) and in group SIL (42%, 42%, and 42%), and reduced in group PLC (26%, 38%, and 27%). In conclusion, fascicular silicone tubulization allowed the highest degree of accuracy but the lowest recovery, whereas resorbable PLC guides provided for the best balance between amount and accuracy of reinnervation after nerve resection.

Ectopic endplates induce localized changes in skeletal muscle ultrastructure.

To investigate the processes by which motoneurons control protein synthesis, and thus the ultrastructure of the muscle fibers they innervate, ectopic endplates were induced to form on adult mouse skeletal muscle fibers by transplantation of a foreign nerve onto the muscle. In the dually innervated muscle fibers thus created, we examined two ultrastructural parameters that correlate with the expression of distinct isoforms of the myofibrillar proteins alpha-actinin and titin, specifically, Z-line width and sarcomere length. It was found that Z-lines were significantly thinner (98 vs. 128 nm) and sarcomeres were significantly shorter (1.69 vs. 2.06 microm) near the ectopic than near the original endplates. Thus, ectopic endplate formation on adult skeletal muscle fibers induces a localized alteration in myofibrillar morphology. These results may help to elucidate the role played by motoneurons in the determination and maintenance of muscle fiber properties and the processes that occur following muscle reinnervation after nerve injury.

Migration of LHRH neurons into the spinal cord: evidence for axon-dependent migration from the transplanted chick olfactory placode.

In the chick embryo, luteinizing hormone-releasing hormone (LHRH) neurons originate in the olfactory placode and migrate along the olfactory nerve to the forebrain. In previous studies, we demonstrated that LHRH neurons followed the trigeminal nerve when the olfactory nerve was physically interrupted. To examine whether LHRH neurons possess the capacity to migrate along the different type of axons, the olfactory placode was transplanted into the base of the forelimb. Three to five days after the transplantation, LHRH neurons were detectable in the spinal nerve, the dorsal root ganglion, the sympathetic ganglion and the spinal cord. Double or triple labelling studies for LHRH, somatostatin and/or axonin-1 showed that LHRH neurons entered the spinal nerve in contact with the olfactory axons, which are specifically immunoreactive to somatostatin. Migrating LHRH neurons continued to associate closely with the olfactory axons in the spinal nerve. However, some LHRH neurons often migrated along with the axonin-1 positive spinal sensory axons, maintaining a distance from the olfactory axons. Furthermore, a few LHRH neurons were observed in the ventral root and the ventral funiculus independent of olfactory axons. As LHRH neurons were observed in the motor component of the spinal nerve, it is probable that LHRH neurons also invaded the spinal cord using the motor axons as a guiding substrate for their migration. These results suggest that the migration mode of LHRH neurons is axon dependent in the peripheral region, however, chemical identity with regard to axonal substrate choice for migration was not specified in the present study.

Development of a bioartificial nerve graft. II. Nerve regeneration in vitro.

A promising alternative for the repair of peripheral nerve injuries is the bioartificial nerve graft, or BNG, comprised of a tubular conduit preseeded with Schwann cells, which are an effective substrate for enhancing nerve regeneration. The physical properties of the conduit, porosity and wall thickness, as well as the Schwann cell seeding density, were tested for their effect on axon growth using rat dorsal root ganglia. These parameters can influence the amount of nutrients and growth factors made available to the neural tissue. Results show that a greater wall thickness and lower porosities have a detrimental effect on the growth of the axons. Over a four week period, axons extended 3.2 mm for the optimum case (DeltaR = 0.82 mm, epsilon = 0.75) compared to 1.8 and 1.6 mm for a lower porosity (0.55) and a greater wall thickness (1.4 mm), respectively. A maximum in the growth rate occurs at a porosity of 75% for Schwann cell seeded conduits but not for unseeded ones. When compared to mass transfer predictions, the results suggest that, at higher porosities, more growth factors diffuse out of the conduit, while at low porosities there is competition for nutrients. Increasing the Schwann cell seeding density enhances growth but also leads to an increase in the number of axons along the length of the conduit. This is indicative of branching of the axons, which requires additional resources to maintain and can lead to painful neuroma formation. Wall thickness and porosity were found not to have any significant effect on the axon number sprouting from the dorsal root ganglia and the mean diameter (p > 0.05). Considerations need to be made, not just on the polymer used, but also on its porosity, wall thickness, and Schwann cell seeding density. These parameters can be adjusted to create a bioartificial nerve graft that provides the optimal environment for nerve growth.