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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Lamina propria and olfactory bulb ensheathing cells exhibit differential integration and migration and promote differential axon sprouting in the lesioned spinal cord.

Olfactory bulb-derived (central) ensheathing cell (OB OEC) transplants have shown significant promise in rat models of spinal cord injury, prompting the use of lamina propria-derived (peripheral) olfactory ensheathing cells (LP OECs) in both experimental and clinical trials. Although derived from a common embryonic precursor, both sources of OECs reside in different nervous system compartments postnatally, and their ability to promote regeneration and efficacy after transplantation may differ depending on both their source and mode of transplantation. Here, we have purified green fluorescent protein-expressing LP and OB OECs, assayed their biological differences in vitro, and transplanted them acutely either directly into or rostral and caudal to a dorsolateral funiculus crush. LP and OB OECs exhibit multiple morphological and antigenic similarities in vitro, and, after transplantation, they both attenuate lesion and cavity formation and promote angiogenesis, endogenous Schwann cell infiltration, and axonal sprouting. However, an increased mitotic rate and migratory ability of LP OECs in vitro was reflected in vivo by their superior ability to migrate within the spinal cord, reduce cavity formation and lesion size, and differentially stimulate outgrowth of axonal subpopulations compared with OB OECs. An undesired behavior (autotomy) was also significantly enhanced by LP OEC, over OB OEC, transplantation. These results suggest that LP and OB OECs exhibit intrinsic biological differences that, after transplantation into the lesioned CNS, result in differences in postlesion spinal cord neuropathology and anatomical and behavioral regeneration outcomes that also vary depending on direct versus rostrocaudal transplantation.

Cross-reinnervation changes the expression patterns of the monocarboxylate transporters 1 and 4: An experimental study in slow and fast rat skeletal muscle.

The monocarboxylate transporters 1 and 4 are expressed in brain as well as in skeletal muscle and play important roles in the energy metabolism of both tissues. In brain, monocarboxylate transporter 1 occurs in astrocytes, ependymocytes, and endothelial cells while monocarboxylate transporter 4 appears to be restricted to astrocytes. In muscle, monocarboxylate transporter 1 is enriched in oxidative muscle fibers whereas monocarboxylate transporter 4 is expressed in all fibers, with the lowest levels in oxidative fiber types. The mechanisms regulating monocarboxylate transporter 1 and monocarboxylate transporter 4 expression are not known. We hypothesized that the expression of these transporters would be sensitive to long term changes in metabolic activity level. This hypothesis can be tested in rat skeletal muscle, where permanent changes in activity level can be induced by cross-reinnervation. We transplanted motor axons originally innervating the fast-twitch extensor digitorum longus muscle to the slow-twitch soleus muscle and vice versa. Four months later, microscopic analysis revealed transformation of muscle fiber types in the cross-reinnervated muscles. Western blot analysis showed that monocarboxylate transporter 1 was increased by 140% in extensor digitorum longus muscle and decreased by 30% in soleus muscle after cross-reinnervation. In contrast, cross-reinnervation induced a 62% decrease of monocarboxylate transporter 4 in extensor digitorum longus muscle and a 1300% increase in soleus muscle. Our findings show that cross-reinnervation causes pronounced changes in the expression levels of monocarboxylate transporter 1 and monocarboxylate transporter 4, probably as a direct consequence of the new pattern of nerve impulses. The data indicate that the mode of innervation dictates the expression of monocarboxylate transporter proteins in the target cells and that the change in monocarboxylate transporter isoform profile is an integral part of the muscle fiber transformation that occurs after cross-reinnervation. Our findings support the hypothesis that the expression of monocarboxylate transporter 1 and monocarboxylate transporter 4 in excitable tissues is regulated by activity.

Development of transplantable nervous tissue constructs comprised of stretch-grown axons.

Pursuing a new approach to nervous system repair, fasciculated axon tracts grown in vitro were developed into nervous tissue constructs designed to span peripheral nerve or spinal cord lesions. We optimized the newfound process of extreme axon stretch growth to maximize the number and length of axon tracts, reach an unprecedented axon growth-rate of 1cm/day, and create 5cm long axon tracts in 8 days to serve as the core component of a living nervous tissue construct. Immunocytochemical analysis confirmed that elongating fibers were axons, and that all major cytoskeletal constituents were present across the stretch-growth regions. We formed a transplantable nervous tissue construct by encasing the elongated cells in an 80% collagen hydrogel, removing them from culture, and inserting them into a synthetic conduit. Alternatively, we induced axon stretch growth directly on a surgical membrane that could be removed from the elongation device, and formed into a cylindrical construct suitable for transplant. The ability to rapidly create living nervous tissue constructs that recapitulates the uniaxial orientations of the original nerve offers an unexplored and potentially complimentary direction in nerve repair. Ideally, bridging nerve damage with living axon tracts may serve to establish or promote new functional connections.

Engineering novel spinal circuits to promote recovery after spinal injury.

We have developed an innovative way to establish a functional bridge around a spinal lesion. We disconnected the T13 nerve from its muscle targets, leaving the proximal end intact. The cut end was inserted either into an intact spinal cord, to assess regeneration of T13 axons into the cord and synapse formation with spinal neurons, or caudal to a hemisection at L2/3, to assess restoration of function below the injury. Four to 28 weeks later, anterograde tracers indicated that axons from the inserted T13 nerve regenerated into the ventral horn, the intermediate zone, and dorsal horn base, both in intact and hemisected animals. Antibodies to cholinergic markers showed that many regenerating axons were from T13 motoneurons. Electrical stimulation of the T13 nerve proximal to the insertion site 4 weeks or more after insertion into the intact cord evoked local field potentials in the intermediate zone and ventral horn, which is where T13 axons terminated. Stimulation of T13 in 71% of the animals (8 hemisected, 7 intact) evoked contraction of the back or leg muscles, depending on the level of insertion. Animals in which T13 was inserted caudal to hemisection had significantly less spasticity and muscle wasting and greater mobility at the hip, knee, ankle, and digits in the ipsilateral hindlimb than did animals with a hemisection only. Thus, T13 motor axons form novel synapses with lumbosacral motor circuits. Because the T13 motor neurons retain their connections to the brain, these novel circuits might restore voluntary control to muscles paralyzed below a spinal lesion.

A novel DNA enzyme reduces glycosaminoglycan chains in the glial scar and allows microtransplanted dorsal root ganglia axons to regenerate beyond lesions in the spinal cord.

CNS lesions induce production of ECM molecules that inhibit axon regeneration. One major inhibitory family is the chondroitin sulfate proteoglycans (CSPGs). Reduction of their glycosaminoglycan (GAG) chains with chondroitinase ABC leads to increased axon regeneration that does not extend well past the lesion. Chondroitinase ABC, however, is unable to completely digest the GAG chains from the protein core, leaving an inhibitory "stub" carbohydrate behind. We used a newly designed DNA enzyme, which targets the mRNA of a critical enzyme that initiates glycosylation of the protein backbone of PGs, xylosyltransferase-1. DNA enzyme administration to TGF-beta-stimulated astrocytes in culture reduced specific GAG chains. The same DNA enzyme applied to the injured spinal cord led to a strong reduction of the GAG chains in the lesion penumbra and allowed axons to regenerate around the core of the lesion. Our experiments demonstrate the critical role of PGs, and particularly those in the penumbra, in causing regeneration failure in the adult spinal cord.

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.

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.

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.