Side-to-side supercharging nerve allograft enhances neurotrophic potential.

INTRODUCTION: Critical limitations of processed acellular nerve allograft (PNA) are linked to Schwann cell function. Side-to-side bridge grafting may enhance PNA neurotrophic potential. METHODS: Sprague-Dawley rats underwent tibial nerve transection and immediate repair with 20-mm PNA (n = 33) or isograft (ISO; n = 9) or 40-mm PNA (n = 33) or ISO (n = 9). Processed acellular nerve allograft groups received zero, one, or three side-to-side bridge grafts between the peroneal nerve and graft. Muscle weight, force generation, and nerve histomorphology were tested 20 weeks after repair. Selected animals underwent neuron back labeling with fluorescent dyes. RESULTS: Inner axon diameters, g-ratios, and axon counts were smaller in the distal vs proximal aspect of each graft (P < .05). Schwann cell counts were greater, with a lower proportion of senescent cells for groups with bridges (P < .05). Retrograde labeling demonstrated that 6.6% to 17.7% of reinnervating neurons were from the peroneal pool. DISCUSSION: Bridge grafting positively influenced muscle recovery and Schwann cell counts and senescence after long PNA nerve reconstruction.

Clinical Outcomes of Nerve Transfers in Peroneal Nerve Palsy: A Systematic Review and Meta-Analysis.

BACKGROUND: Given the unsatisfactory outcomes with traditional treatments, there is growing interest in nerve transfers to reestablish ankle dorsiflexion in peroneal nerve palsy. The objective of this work was to perform a systematic review and meta-analysis of the primary literature to assess the effectiveness of nerve transfer surgery in restoring ankle dorsiflexion in patients with peroneal nerve palsy. METHODS: Methodology was registered with PROSPERO, and PRISMA guidelines were followed. MEDLINE, EMBASE, and the Cochrane Library were systematically searched. English studies investigating outcomes of nerve transfers in peroneal nerve palsy were included. Two reviewers completed screening and extraction. Methodological quality was evaluated with Newcastle-Ottawa Scale. RESULTS: Literature search identified 108 unique articles. Following screening, 14 full-text articles were reviewed. Four retrospective case series met inclusion criteria for meta-analysis. Overall, 41 patients underwent nerve transfer for peroneal nerve palsy. The mean age of the patients was 36.1 years, mean time to surgery was 6.3 months, and the mean follow-up period was 19.0 months. Donor nerve was either tibial (n = 36) or superficial peroneal branches/fascicles (n = 5). Recipient nerve was either deep peroneal (n = 24) or tibialis anterior branch (n = 17). Postoperative ankle dorsiflexion strength demonstrated a bimodal distribution with a mean Medical Research Council of 2.1. There were no significant differences in dorsiflexion strength between injury sites (p = 0.491), injury mechanisms (p = 0.125), donor (p = 0.066), or recipient nerves (p = 0.496). There were no significant correlations between dorsiflexion strength and patient age (p = 0.094) or time to surgery (p = 0.493). CONCLUSIONS: There is variability in dorsiflexion strength following nerve transfer in peroneal nerve palsy, whereby there appear to be responders and non-responders. Further studies are needed to better define appropriate patient selection and the role of nerve transfers in the management of peroneal nerve palsy.

Polyethylene glycol treated allografts not tissue matched nor immunosuppressed rapidly repair sciatic nerve gaps, maintain neuromuscular functions, and restore voluntary behaviors in female rats.

Many publications report that ablations of segments of peripheral nerves produce the following unfortunate results: (1) Immediate loss of sensory signaling and motor control; (2) rapid Wallerian degeneration of severed distal axons within days; (3) muscle atrophy within weeks; (4) poor behavioral (functional) recovery after many months, if ever, by slowly-regenerating (∼1mm/d) axon outgrowths from surviving proximal nerve stumps; and (5) Nerve allografts to repair gap injuries are rejected, often even if tissue matched and immunosuppressed. In contrast, using a female rat sciatic nerve model system, we report that neurorrhaphy of allografts plus a well-specified-sequence of solutions (one containing polyethylene glycol: PEG) successfully addresses each of these problems by: (a) Reestablishing axonal continuity/signaling within minutes by nonspecific ally PEG-fusing (connecting) severed motor and sensory axons across each anastomosis; (b) preventing Wallerian degeneration by maintaining many distal segments of inappropriately-reconnected, PEG-fused axons that continuously activate nerve-muscle junctions; (c) maintaining innervation of muscle fibers that undergo much less atrophy than otherwise-denervated muscle fibers; (d) inducing remarkable behavioral recovery to near-unoperated levels within days to weeks, almost certainly by CNS and PNS plasticities well-beyond what most neuroscientists currently imagine; and (e) preventing rejection of PEG-fused donor nerve allografts with no tissue matching or immunosuppression. Similar behavioral results are produced by PEG-fused autografts. All results for Negative Control allografts agree with current neuroscience data 1-5 given above. Hence, PEG-fusion of allografts for repair of ablated peripheral nerve segments expand on previous observations in single-cut injuries, provoke reconsideration of some current neuroscience dogma, and further extend the potential of PEG-fusion in clinical practice.

Targeted mesenchymal stem cell and vascular endothelial growth factor strategies for repair of nerve defects with nerve tissue implanted autogenous vein graft conduits.

Peripheral nerve gaps exceeding 1 cm require a bridging repair strategy. Clinical feasibility of autogenous nerve grafting is limited by donor site comorbidity. In this study we investigated neuroregenerative efficacy of autogenous vein grafts implanted with tissue fragments from distal nerve in combination with vascular endothelial growth factor (VEGF) or mesenchymal stem cells (MSCs) in repair of rat peripheral nerve defects. Six-groups of Sprague-Dawley rats (n = 8 each) were evaluated in the autogenous setting using a 1.6 cm long peroneal nerve defect: Empty vein graft (group 1), Nerve graft (group 2), Vein graft and nerve fragments (group 3), Vein graft and nerve fragments and blank microspheres (group 4), Vein graft and nerve fragments and VEGF microspheres (group 5), Vein graft and nerve fragments and MSCs (group 6). Nerve fragments were derived from distal segment. Walking track analysis, electrophysiology and nerve histomorphometry were performed for assessment. Peroneal function indices (PFI), electrophysiology (amplitude) and axon count results for group 2 were -9.12 ± 3.07, 12.81 ± 2.46 mV, and 1697.88 ± 166.18, whereas the results for group 5 were -9.35 ± 2.55, 12.68 ± 1.78, and 1566 ± 131.44, respectively. The assessment results did not reveal statistical difference between groups 2 and 5 (P > 0.05). The best outcomes were seen in group 2 and 5 followed by group 6. Compared to other groups, poorest outcomes were seen in group 1 (P ≤ 0.05). PFI, electrophysiology (amplitude) and axon count results for group 1 were -208.82 ± 110.69, 0.86 ± 0.52, and 444.50 ± 274.03, respectively. Vein conduits implanted with distal nerve-derived nerve fragments improved axonal regeneration. VEGF was superior to MSCs in facilitating nerve regeneration. © 2015 Wiley Periodicals, Inc. Microsurgery 36:578-585, 2016.

Adipose-derived stem cells protect against endoneurial cell death: Cell therapy for nerve autografts.

BACKGROUND: One of the major problems with nerve grafts is that the survival of a graft segment, including endoneurial Schwann cells (SCs), is uncertain. We investigated whether the survival of nerve grafts is improved when adipose-derived stem cells (ASCs) are incorporated into the grafts. METHODS: To examine the cell-protective effects of ASCs on SCs in vitro, we used an indirect coculture system. In vivo effects of the incorporation of ASCs into grafts were examined using a graft model in the rat common peroneal nerve. Grafts were entubulated to isolate them from the surrounding tissues, mimicking the clinical conditions of a poorly vascularized recipient bed. Thirty-six Lewis rats were divided into three groups, i.e., nerve graft only, entubulated nerve graft, and entubulated nerve graft + ASC transplantation. In each group, four rats and eight rats were used for short-term (10 days) and long-term (12 weeks) follow-up study, respectively. RESULTS: After 24 hours of serum deprivation, the numbers of 7-aminoactinomycin D, and TUNEL-positive SCs significantly decreased when indirectly cocultured with ASCs (P < 0.01). When ASCs were transplanted to the epineurial layer of the grafts, the number of endoneurial TUNEL-positive cells decreased significantly, as compared with grafts without ASCs, at 10 days postoperatively (P < 0.05). Postoperative walking track analysis showed that the ASC-transplanted grafts showed significantly faster function recovery, as compared with grafts without ASCs (P < 0.05). CONCLUSION: These results suggest that nerve autografts + ASC therapy could offer a new approach to obtaining optimal outcomes after peripheral nerve injury.

Functional recovery after repair of peroneal nerve gap using different collagen conduits.

BACKGROUND: Currently, autologous nerve implantation to bridge a long nerve gap presents the greatest regenerative performance in spite of substantial drawbacks. In this study, we evaluate the effect of two different collagen conduits bridging a peroneal nerve gap. METHODS: Rats were divided into four groups: (1) the gold standard group, in which a 10-mm-long nerve segment was cut, reversed, and reimplanted between the nerve stumps; (2) the CG-I/III group, in which a type I/III collagen conduit bridged the gap; (3) the CG-I, in which a type I collagen conduit was grafted; and (4) the sham group, in which a surgery was performed without injuring the nerve. Peroneal Functional Index and kinematics analysis of locomotion were performed weekly during the 12 weeks post-surgery. At the end of the protocol, additional electrophysiological tests, muscular weight measurements, axon counting, and g-ratio analysis were carried out. RESULTS: Functional loss followed by incomplete recovery was observed in animals grafted with collagen conduits. At 12 weeks post-surgery, the ventilatory rate of the CG-I group in response to exercise was similar to the sham group, contrary to the CG-I/III group. After KCl injections, an increase in metabosensitive afferent-fiber activity was recorded, but the response stayed incomplete for the collagen groups compared to the sham group. Furthermore, the CG-I group presented a higher number of axons and seemed to induce a greater axonal maturity compared to the CG-I/III group. CONCLUSIONS: Our results suggest that the grafting of a type I collagen conduit may present slight better prospects than a type I/III collagen conduit.

Single versus double end-to-side nerve grafts in rats.

PURPOSE: Although the end-to-side nerve repair technique has been used clinically, it has not yet produced consistent motor and sensory recovery in patients. The aim of this study was to investigate whether end-to-side double nerve grafts display more axonal regeneration compared with a single nerve graft in a rat lower limb preparation. METHODS: The lower limbs of 96 Wister rats were used in experiments comparing single and double end-to-side nerve grafts. Left peroneal nerves were harvested and grafted between the right peroneal and tibial nerves. A single graft was attached end-to-side to the peroneal and tibial nerves through an epineural window (single graft group, n = 24). Two grafts were performed in the same manner in the double graft group (n = 24). The peroneal nerve was exposed in positive controls (n = 24) and no graft was performed in negative controls (n = 24). We recorded action potentials and moist weights of the left tibialis anterior muscle at each time point. Fluoro-Gold-labeled (Fluorochrome, Denver, CO) dorsal root ganglion neurons from L1 to L6 were counted using fluorescence microscopy and compared among the 4 groups. RESULTS: In both single and double groups, the amplitude and the tibialis anterior muscle weight increased significantly compared with negative controls but remained lower than those measured in positive controls. There was no significant difference between single and double groups. In Fluoro-Gold-labeled neurons, there was also no significant difference between single and double groups. CONCLUSIONS: The study showed that regeneration of motor and sensory nerve fibers was possible using 2 end-to-side nerve grafts. However, there was no significant difference between single and double grafts. This might suggest a therapeutic limitation of nerve transplants using 2 end-to-side nerve grafts. CLINICAL RELEVANCE: Double end-to-side repair attracts both motor and sensory axons, and this results in a medium degree of recovery of function; however, double end-to-side nerve grafting does not appear to offer any advantage over a single end-to-side graft.

Neurotrophin-4/5 is implicated in the enhancement of axon regeneration produced by treadmill training following peripheral nerve injury.

The role of neurotrophin-4/5 (NT-4/5) in the enhancement of axon regeneration in peripheral nerves produced by treadmill training was studied in mice. Common fibular nerves of animals of the H strain of thy-1-YFP mice, in which a subset of axons in peripheral nerves is marked by the presence of yellow fluorescent protein, were cut and surgically repaired using nerve grafts from non-fluorescent mice. Lengths of profiles of fluorescent regenerating axons were measured using optical sections made through whole mounts of harvested nerves. Measurements from mice that had undergone 1 h of daily treadmill training at modest speed (10 m/min) were compared with those of untrained (control) mice. Modest treadmill training resulted in fluorescent axon profiles that were nearly twice as long as controls at 1, 2 and 4 week survival times. Similar enhanced regeneration was found when cut nerves of wild type mice were repaired with grafts from NT-4/5 knockout mice or grafts made acellular by repeated freezing/thawing. No enhancement was produced by treadmill training in NT-4/5 knockout mice, irrespective of the nature of the graft used to repair the cut nerve. Much as had been observed previously for the effects of brief electrical stimulation, the effects of treadmill training on axon regeneration in cut peripheral nerves are independent of changes produced in the distal segment of the cut nerve and depend on the promotion of axon regeneration by changes in NT-4/5 expression by cells in the proximal nerve segment.

Superficial peroneal nerve accessory artery (SPNAA) flap for head and neck reconstruction: A cadaveric anatomical study and retrospective case series review.

BACKGROUND: Several different flaps can reconstruct intraoral defects or lower limb deficits after free fibula osteo-cutaneous flap harvesting for jaw reconstructions. However, commonly used options may not be available for various reasons and can be associated with significant morbidity. We hypothesized that flaps supplied by the superficial peroneal nerve accessory artery (SPNAA) could be a viable alternative reconstructive option. METHODS: We describe the SPNAA's anatomy using 20 human cadaveric leg dissections and report eight cases involving SPNAA-based perforator flap reconstructions (six propeller flaps and two free flaps) in a retrospective case series. Patient-specific baseline variables and intraoperative and postoperative outcomes are described. RESULTS: Cadaveric dissection suggests that the location of the SPNAA is reliable but its origin varies, with 40% (N = 8) of SPNAAs being of type I origin, 20% type II (N = 4), and 40% (N = 8) type III in our series. All reconstructions were successful. No intraoperative complications occurred during propeller or free-flap reconstructions. No flap failures occurred. One propeller reconstruction showed distal superficial skin necrosis and one donor site wound dehisced; both were successfully managed conservatively. No other short-term or long-term complications occurred. CONCLUSIONS: Flaps based on SPNAA perforators appear effective, reliable, and safe reconstructive methods for covering fibula osteocutaneous donor site defects and for intraoral reconstructions. Controlled trials are required to compare its effectiveness and safety with other reconstructive methods.

Anterior transposition of the superficial peroneal nerve branch during the internal fixation of the lateral malleolus.

BACKGROUND: Because of the anatomic variations of the superficial peroneal nerve (SPN), it has long been stressed that caution should be exercised at the time of open reduction and internal fixation (ORIF) of the lateral malleolus. METHODS: Blair and Botte described type B intermediate dorsal cutaneous nerve (IDCN) in which the SPN penetrates crural fascia posterior to the fibula about 5 cm proximal to the joint and crosses the lateral border of the fibulae. We hypothesized that the type B IDCN is especially vulnerable to direct surgical injury if present and the anterior transposition of this nerve may decrease the incidence of symptoms related to the SPN injury. Fifty-three ankle fractures in 53 adult patients treated by the ORIF of lateral malleolus using the lateral approach between the periods from May 2001 to December 2006 were included. Intraoperative documentation about the presence of the type B IDCN variant at the surgical field was performed, and preoperative and postoperative sensory changes were carefully evaluated. RESULTS: We encountered Blair type B variant in 7 cases (12%). The IDCN was carefully dissected and transposed anteriorly before the plating. One of these seven patients had sensory deficit preoperatively, and it was recovered spontaneously 5 months after operation. There was one patient whose IDCN was inadvertently severed, and it was repaired. At the time of last follow-up, only partial recovery of the sensory deficit was noted. Five of seven patients did not show any neurologic deficit with anterior transposition. CONCLUSION: Recognition and anterior transposition of the type B IDCN could reduce the incidence of the SPN nerve injuries during the ORIF of the lateral malleolar fractures.

Repair of whole rabbit facial nerve defects using facial nerve allografts.

PURPOSE: To investigate the feasibility of repairing whole facial nerve defects with chemically extracted acellular whole facial allografts nerves and its effect on motor conductivity recovery. MATERIALS AND METHODS: Whole nerve defects (branches and trunk) were made in 4 rabbit groups (n = 18), and the nerve defect was bridged using 1) acellular facial nerve allografts, 2) facial nerve isografts, 3) acellular peroneal nerve allografts, and 4) peroneal nerve isografts. Six months later, cell morphology, nerve microbeam distribution, angiogenesis, and collagen were observed in the distal and center of the grafts with special trichrome staining. The regenerated nerve fibers and Schwann cells in the anastomosis site were immunohistochemically stained. Nerve axon numbers and passing rates were analyzed with computer-captured images. The regenerated nerve ultrastructure was analyzed by transmission electron microscopy. RESULTS: Regenerated nerve fibers and vessels were found in the grafts, with no differences between groups A and B. Groups C and D had poor nerve continuity with little vascular regeneration. The distal segments of nerve transplants in groups A and B showed strong positive neurofilament staining, higher than in groups C and D. In groups A and B, many long spindle-shaped Schwann cells proliferated longitudinally in the nerve transplant, but less in groups C and D. Myelinated nerve fibers were found in the distal facial nerve. There were no differences between groups A and B in fiber number and myelin sheath thickness, which were much lower than normal, whereas little myelin sheath regeneration was observed in groups C and D. CONCLUSION: Chemically extracted acellular whole facial nerve allografts are feasible for repairing whole facial nerve defects.

Comparison of the performance of chronically versus freshly denervated autograft in nerve repair.

PURPOSE: Graft choice is one of the few variables over which the surgeon has control when reconstructing nerve gaps. Because repair of chronically denervated nerves generally yields inferior recovery, we hypothesized that the use of chronically denervated nerve tissue as a graft source may compromise axonal regeneration and clinical results. METHODS: A total of 45 immature female Sprague-Dawley rats underwent transection of one peroneal nerve before being divided into 3 experimental groups: group A (n = 15) had acutely denervated nerve graft, group B (n = 15) had 2-month denervated nerve graft, and group C (n = 15) had 4-month denervated nerve graft. We included 10 additional rats as a sham group. After 2 months, groups A and B underwent removal of 1 cm of the contralateral peroneal nerve. For group A, this section of nerve was immediately sutured back in place to serve as a model for acute denervation. For group B, the defect was repaired with a 1-cm graft from the distal stump of the previously transected (denervated) peroneal nerve. Group C underwent the same procedure as group B, but after an additional 2 months. After 8 weeks of regeneration time, the 3 experimental groups and the sham group underwent testing. We assessed twitch contraction forces of the reinnervated extensor digitorum longus before we harvested the muscle belly for morphologic measurements. Histological nerve tissue evaluation assessed axonal regeneration. RESULTS: We detected no statistical differences for mean muscle contraction strengths between the experimental groups; nevertheless, the reinnervated extensor digitorum longus muscle bellies from the 4-month denervated nerve graft group were statistically smaller than muscles from the other 2 experimental groups (p < .05). Axon counts decreased, whereas axon diameters increased in direct correlation with the length of time of graft denervation (p < .05). No difference in axon myelination was found between experimental groups. CONCLUSIONS: Prolonged denervation of nerve graft material compromised both axon and reinnervated muscle recovery in this rodent model.

Surgical anatomy of sensory portion of superficial fibular nerve for harvesting nerve grafts from fetuses.

The use of superficial fibular nerve (Sfn) as a potential donor nerve in nerve grafting has been introduced. The limited availability of donor nerves has paved the way for nerve allografting. We studied the sensory portion of Sfn in 60 limbs from 30 fetuses. Three distinct patterns of the nerve were designated as Types 1, 2, and 3 by us. Type 1 (66.67%) comprised Sfn piercing fascia cruris then branching into Mdn and Idn. Type 2 (21.67%) was a pattern where Sfn penetrated deep fascia then continued undivided over the dorsum of foot. Type 3 (11.67%) was where Mdn and Idn penetrated deep fascia independently. The study provided quantitative measurement data of the sensory portion of Sfn and its branching nerves with respect to osseous landmarks like the head of fibula and the malleoli. Such data may be of help in defining nerve segments suitable for harvesting in nerve grafts from fetuses.

Nerve Repair and Orthodromic and Antidromic Nerve Grafts: An Experimental Comparative Study in Rabbit.

PURPOSE: Although many surgeons have anecdotally described reversing the polarity of the autograft with the intent of improving regeneration, the optimal orientation of the autogenous nerve graft remains controversial. The aim of this study was to compare (1) the outcomes of orthodromic and antidromic nerve grafts to clarify the effect of nerve graft polarity and (2) the outcome of either form of nerve grafts with that of nerve repair. METHODS: In 14 of the 26 rabbits used in this study, a 1 cm defect was made in the tibial nerve. An orthodromic nerve graft on one side and an antidromic nerve graft on the other were performed using a 1.2 cm long segment of the peroneal nerve. In the remaining 12 rabbits, the tibial nerve was transected completely and then repaired microscopically on one side but left untreated on the other. Electrophysiologic studies were performed in all animals at 8 weeks after surgery, and the sciatic nerves were harvested. RESULTS: Compound motor action potential was visible in all rabbits treated by nerve repair but in only half of the rabbits treated by nerve graft. There was no significant difference in the compound motor action potential, nerve conduction velocity, or total number of axons between the orthodromic and antidromic nerve graft groups. However, in both groups, the outcome was significantly poorer than that of the nerve repair group. CONCLUSION: There was no significant difference by electromyographic or histologic evaluation between orthodromic and antidromic nerve grafts. Direct nerve repair with moderate tension may be a more effective treatment than nerve grafting.

A single session of brief electrical stimulation enhances axon regeneration through nerve autografts.

Nerve graft reconstruction of gap defects may result in poor clinical outcomes, particularly with long regeneration distances. Electrical stimulation (ES) of nerves may improve outcomes in such patients. A single session of ES at 20 Hz for 1 h significantly enhances axon regeneration in animals and human subjects after nerve crush or nerve transection and repair. The objectives of this study were to evaluate if ES enhances axon regeneration through nerve grafts and if there is added benefit of a second, delayed session of ES (serial ES) on axon regeneration as compared to a single session only of ES. In female rats, a gap defect was created in the hindlimb common peroneal (CP) nerve and immediately reconstructed with a 10 mm nerve autograft (Experiment 1) or a 20 mm nerve autograft (Experiment 2). In Experiment 1, rats were randomized to 1 h of CP nerve ES or sham stimulation. In Experiment 2, rats were randomized to control (sham ES + sham ES), single ES (ES + sham ES), or serial ES (ES + ES), which consisted of an initial 1 h session of either ES or sham stimulation of the CP nerve, followed by a second 1 h session of ES or sham stimulation of the CP nerve 4 weeks later. In both experiments, after a 6 week period of nerve regeneration, CP neurons that had regenerated axons distal to the autograft were retrograde labelled for enumeration, and the CP nerve distal to the autograft was harvested for histomorphometry. In Experiment 1, rats that received CP nerve ES had statistically significantly more motor (p < .05) and sensory (p < .05) neurons that regenerated axons distal to the 10 mm nerve autograft, with more myelinated axons on histomorphometry (p < .001). Similarly, in Experiment 2, significantly more motor (p < .01) and sensory (p < .05) neurons regenerated axons distal to the 20 mm nerve autograft after a single session or two sessions of CP nerve ES. There was no significant difference in the number of regenerated motor or sensory neurons between rats with 20 mm CP nerve autografts receiving either one or two sessions of CP nerve ES (p > .05). In conclusion, a single session of ES enhances axon regeneration following nerve autografting with no added effect of a second, delayed session of ES. These findings support previous studies in animals and humans of the robust effect of a single session of ES in promoting nerve regeneration following injury and repair.

Regeneration of adult rat sensory and motor neuron axons through chimeric peroneal nerve grafts containing donor Schwann cells engineered to express different neurotrophic factors.

Large peripheral nerve (PN) defects require bridging substrates to restore tissue continuity and permit the regrowth of sensory and motor axons. We previously showed that cell-free PN segments repopulated ex vivo with Schwann cells (SCs) transduced with lentiviral vectors (LV) to express different growth factors (BDNF, CNTF or NT-3) supported the regeneration of axons across a 1 cm peroneal nerve defect (Godinho et al., 2013). Graft morphology, the number of regrown axons, the ratio of myelinated to unmyelinated axons, and hindlimb locomotor function differed depending on the growth factor engineered into SCs. Here we extend these observations, adding more LVs (expressing GDNF or NGF) and characterising regenerating sensory and motor neurons after injection of the retrograde tracer Fluorogold (FG) into peroneal nerve distal to grafts, 10 weeks after surgery. Counts were also made in rats with intact nerves and in animals receiving autografts, acellular grafts, or grafts containing LV-GFP transduced SCs. Counts and analysis of FG positive (+) DRG neurons were made from lumbar (L5) ganglia. Graft groups contained fewer labeled sensory neurons than non-operated controls, but this decrease was only significant in the LV-GDNF group. These grafts had a complex fascicular morphology that may have resulted in axon trapping. The proportion of FG+ sensory neurons immunopositive for calcitonin-gene related peptide (CGRP) varied between groups, there being a significantly higher percentage in autografts and most neurotrophic factor groups compared to the LV-CNTF, LV-GFP and acellular groups. Furthermore, the proportion of regenerating isolectin B4+ neurons was significantly greater in the LV-NT-3 group compared to other groups, including autografts and non-lesion controls. Immunohistochemical analysis of longitudinal graft sections revealed that all grafts contained a reduced number of choline acetyltransferase (ChAT) positive axons, but this decrease was significant only in the GDNF and NT-3 graft groups. We also assessed the number and phenotype of regrowing lumbar FG+ motor neurons in non-lesioned animals, and in rats with autografts, acellular grafts, or in grafts containing SCs expressing GFP, CNTF, NGF or NT-3. The overall number of FG+ motor neurons per section was similar in all groups; however in tissue immunostained for NeuN (expressed in α- but not γ-motor neurons) the proportion of NeuN negative FG+ neurons ranged from about 40-50% in all groups except the NT-3 group, where the percentage was 82%, significantly more than the SC-GFP group. Immunostaining for the vesicular glutamate transporter VGLUT-1 revealed occasional proprioceptive terminals in 'contact' with regenerating FG+ α-motor neurons in PN grafted animals, the acellular group having the lowest counts. In sum, while all graft types supported sensory and motor axon regrowth, there appeared to be axon trapping in SC-GDNF grafts, and data from the SC-NT-3 group revealed greater regeneration of sensory CGRP+ and IB4+ neurons, preferential regeneration of γ-motor neurons and perhaps partial restoration of monosynaptic sensorimotor relays.

Functional Outcome after Reconstruction of a Long Nerve Gap in Rabbits Using Optimized Decellularized Nerve Allografts.

BACKGROUND: Processed nerve allografts are a promising alternative to nerve autografts, providing an unlimited, readily available supply and avoiding donor-site morbidity and the need for immunosuppression. Currently, clinically available nerve allografts do not provide satisfactory results for motor reconstruction. This study evaluated motor recovery after reconstruction of a long nerve gap using a processed nerve allograft and the influence of storage techniques. METHODS: Nerve allografts were decellularized using elastase and detergents and stored at either 4° or -80°C. In 36 New Zealand White rabbits, a 3-cm peroneal nerve gap was repaired with either an autograft (group 1, control) or a cold-stored (group 2) or frozen-stored (group 3) processed nerve allograft. Nerve recovery was evaluated using longitudinal ultrasound measurements, electrophysiology (compound muscle action potentials), isometric tetanic force, wet muscle weight, and histomorphometry after 24 weeks. RESULTS: Longitudinal ultrasound measurements showed that the cold-stored allograft provided earlier regeneration than the frozen-stored allograft. Furthermore, ultrasound showed significantly inferior recovery in group 3 than in both other groups (p < 0.05). Muscle weight and isometric tetanic force showed similar outcomes in the autograft and cold-stored allograft groups [p = 0.096 (muscle weight) and p = 0.286 (isometric tetanic force)], and confirmed the inferiority of the frozen-stored allograft to the autograft [p < 0.01 (muscle weight) and p = 0.02 (isometric tetanic force)]. CONCLUSIONS: Frozen storage of the nerve allograft significantly impairs functional recovery and should be avoided. The cold-stored optimized nerve allograft yields functional recovery similar to the gold standard autograft in the reconstruction of a 3-cm motor nerve defect. Future studies should focus on further improvement of the nerve allograft.

Deoxyribozyme-mediated knockdown of xylosyltransferase-1 mRNA promotes axon growth in the adult rat spinal cord.

In the injured spinal cord, proteoglycans (PGs) within scar tissue obstruct axon growth through their glycosaminoglycan (GAG)-side chains. The formation of GAG-side chains (glycosylation) is catalysed by xylosyltransferase-1 (XT-1). Here, we knocked down XT-1 mRNA using a tailored deoxyribozyme (DNAXTas) and hypothesized that this would decrease the amount of glycosylated PGs and, consequently, promote axon growth in the adult rat spinal cord. A continuous 2-week delivery of DNAXTas near the rostral border of a peripheral nerve graft bridging the transected dorsal columns in the thoracic spinal cord resulted in an 81% decrease in XT-1 mRNA, an average of 1.4-fold reduction in GAG-side chains of chondroitin sulphate or heparan sulphate-PGs and 2.2-fold reduction in neurocan and brevican core proteins in scar tissue. Additionally, compared to control deoxyribozyme, the DNAXTas treatment resulted in a 9-fold increase in length and a 4-fold increase in density of ascending axons growing through the nerve graft and scar tissue present at the rostral spinal cord. Together our data showed that treatment with a deoxyribozyme against XT-1 mRNA decreased the amount of glycosylated PGs and promoted axon growth through scar tissue in the injured spinal cord. The deoxyribozyme approach may become a contributing factor in spinal cord repair strategies.

Novel experimental surgical strategy to prevent traumatic neuroma formation by combining a 3D-printed Y-tube with an autograft.

OBJECTIVE Traumatic neuromas may develop after nerve injury at the proximal nerve stump, which can lead to neuropathic pain. These neuromas are often resistant to therapy, and excision of the neuroma frequently leads to recurrence. In this study, the authors present a novel surgical strategy to prevent neuroma formation based on the principle of centro-central anastomosis (CCA), but rather than directly connecting the nerve ends to an autograft, they created a loop using a 3D-printed polyethylene Y-shaped conduit with an autograft in the distal outlets. METHODS The 3D-printed Y-tube with autograft was investigated in a model of rat sciatic nerve transection in which the Y-tube was placed on the proximal sciatic nerve stump and a peroneal graft was placed between the distal outlets of the Y-tube to form a closed loop. This model was compared with a CCA model, in which a loop was created between the proximal tibial and peroneal nerves with a peroneal autograft. Additional control groups consisted of the closed Y-tube and the extended-arm Y-tube. Results were analyzed at 12 weeks of survival using nerve morphometry for the occurrence of neuroma formation and axonal regeneration in plastic semi-thin sections. RESULTS Among the different surgical groups, the Y-tube with interposed autograft was the only model that did not result in neuroma formation at 12 weeks of survival. In addition, a 13% reduction in the number of myelinated axons regenerating through the interposed autograft was observed in the Y-tube with autograft model. In the CCA model, the authors also observed a decrease of 17% in the number of myelinated axons, but neuroma formation was present in this model. The closed Y-tube resulted in minimal nerve regeneration inside the tube together with extensive neuroma formation before the entrance of the tube. The extended-arm Y-tube model clearly showed that the majority of the regenerating axons merged into the Y-tube arm, which was connected to the autograft, leaving the extended plastic arm almost empty. CONCLUSIONS This pilot study shows that our novel 3D-printed Y-tube model with interposed autograft prevents neuroma formation, making this a promising surgical tool for the management of traumatic neuromas.

Peripheral nerve regeneration through allografts compared with autografts in FK506-treated monkeys.

OBJECT: The clinical use of nerve allografts combined with immunosuppressant therapy has become a genuine possibility that could supersede the classic use of autografts. However, contradictory data have been reported on whether immunosuppressant therapy should be temporarily administered. The purpose of this study was to compare the nerve regeneration obtained using ulnar nerve allografts in nonhuman primates temporarily treated with FK506 (tacrolimus) with that obtained using nerve autografts. METHODS: Four-centimeter nerve autografts or allografts were placed in the distal ulnar motor nerve of eight monkeys. The FK506 was temporarily administered to the animals of the allograft group for 2 months. At periods of 3, 5, and 8 months postsurgery, quantitative electrophysiological recordings were obtained to estimate muscle response. A quantitative analysis of ulnar motor neurons in the spinal cord was performed and axons were counted stereologically. No statistically significant differences were found in the neuronal and axonal counts between autograft and allograft groups at 8 months. The electrophysiological studies showed no differences relative to the amplitude, but the autograft group presented with a greater nerve conduction velocity (NCV). However, no statistically significant differences were found between the number of neurons and distal axonal counts in the two groups. CONCLUSIONS: Nerve regeneration through cold-preserved allografts in a primate model temporarily treated with FK506 was similar to that obtained using nerve autografts, in terms of neuronal and axonal counts. Nevertheless, temporary immunosuppression produced lower NCV when allografts were used, with less maturation of the myelinated fibers, which indicated that a partial rejection had taken place.