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1.  Misdirection of regenerating motor axons after nerve injury and repair in the rat sciatic nerve model 
Experimental neurology  2008;211(2):339-350.
Misdirection of regenerating axons is one of the factors that can explain the poor results often found after nerve injury and repair. In this study, we quantified the degree of misdirection and the effect on recovery of function after different types of nerve injury and repair in the rat sciatic nerve model; crush injury, direct coaptation, and autograft repair. Sequential tracing with retrograde labeling of the peroneal nerve before and 8 weeks after nerve injury and repair was performed to quantify the accuracy of motor axon regeneration. Digital video analysis of ankle motion was used to investigate the recovery of function. In addition, serial compound action potential recordings and nerve and muscle morphometry were performed. In our study, accuracy of motor axon regeneration was found to be limited; only 71% (±4.9%) of the peroneal motoneurons were correctly directed 2 months after sciatic crush injury, 42% (±4.2%) after direct coaptation, and 25% (±6.6%) after autograft repair. Recovery of ankle motion was incomplete after all types of nerve injury and repair and demonstrated a disturbed balance of ankle plantar and dorsiflexion. The number of motoneurons from which axons had regenerated was not significantly different from normal. The number of myelinated axons was significantly increased distal to the site of injury. Misdirection of regenerating motor axons is a major factor in the poor recovery of nerves that innervate different muscles. The results of this study can be used as basis for developing new nerve repair techniques that may improve the accuracy of regeneration.
doi:10.1016/j.expneurol.2007.12.023
PMCID: PMC2967197  PMID: 18448099
Aberrant reinnervation; Accuracy of regeneration; Ankle motion analysis; Double labeling; Sequential retrograde tracing
2.  Sciatic nerve repair with tissue engineered nerve: Olfactory ensheathing cells seeded poly(lactic-co-glygolic acid) conduit in an animal model 
Indian Journal of Orthopaedics  2013;47(6):547-552.
Background and Aim:
Synthetic nerve conduits have been sought for repair of nerve defects as the autologous nerve grafts causes donor site morbidity and possess other drawbacks. Many strategies have been investigated to improve nerve regeneration through synthetic nerve guided conduits. Olfactory ensheathing cells (OECs) that share both Schwann cell and astrocytic characteristics have been shown to promote axonal regeneration after transplantation. The present study was driven by the hypothesis that tissue-engineered poly(lactic-co-glycolic acid) (PLGA) seeded with OECs would improve peripheral nerve regeneration in a long sciatic nerve defect.
Materials and Methods:
Sciatic nerve gap of 15 mm was created in six adult female Sprague-Dawley rats and implanted with PLGA seeded with OECs. The nerve regeneration was assessed electrophysiologically at 2, 4 and 6 weeks following implantation. Histopathological examination, scanning electron microscopic (SEM) examination and immunohistochemical analysis were performed at the end of the study.
Results:
Nerve conduction studies revealed a significant improvement of nerve conduction velocities whereby the mean nerve conduction velocity increases from 4.2 ΁ 0.4 m/s at week 2 to 27.3 ΁ 5.7 m/s at week 6 post-implantation (P < 0.0001). Histological analysis revealed presence of spindle-shaped cells. Immunohistochemical analysis further demonstrated the expression of S100 protein in both cell nucleus and the cytoplasm in these cells, hence confirming their Schwann-cell-like property. Under SEM, these cells were found to be actively secreting extracellular matrix.
Conclusion:
Tissue-engineered PLGA conduit seeded with OECs provided a permissive environment to facilitate nerve regeneration in a small animal model.
doi:10.4103/0019-5413.121572
PMCID: PMC3868134  PMID: 24379458
Olfactory ensheathing cells; poly(lactic-co-glycolic acid); sciatic nerve defect; tissue engineering
3.  Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration 
Background
Nerve conduits provide a promising strategy for peripheral nerve injury repair. However, the efficiency of nerve conduits to enhance nerve regeneration and functional recovery is often inferior to that of autografts. Nerve conduits require additional factors such as cell adhesion molecules and neurotrophic factors to provide a more conducive microenvironment for nerve regeneration.
Methods
In the present study, poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]} (PLGL) was modified by grafting Gly-Arg-Gly-Asp-Gly (RGD peptide) and nerve growth factor (NGF) for fabricating new PLGL-RGD-NGF nerve conduits to promote nerve regeneration and functional recovery. PLGL-RGD-NGF nerve conduits were tested in the rat sciatic nerve transection model. Rat sciatic nerves were cut off to form a 10 mm defect and repaired with the nerve conduits. All of the 32 Wistar rats were randomly divided into 4 groups: group PLGL-RGD-NGF, group PLGL-RGD, group PLGL and group autograft. At 3 months after surgery, the regenerated rat sciatic nerve was evaluated by footprint analysis, electrophysiology, and histologic assessment. Experimental data were processed using the statistical software SPSS 10.0.
Results
The sciatic function index value of groups PLGL-RGD-NGF and autograft was significantly higher than those of groups PLGL-RGD and PLGL. The nerve conduction velocities of groups PLGL-RGD-NGF and autograft were significantly faster than those of groups PLGL-RGD and PLGL. The regenerated nerves of groups PLGL-RGD-NGF and autograft were more mature than those of groups PLGL-RGD and PLGL. There was no significant difference between groups PLGL-RGD-NGF and autograft.
Conclusions
PLGL-RGD-NGF nerve conduits are more effective in regenerating nerves than both PLGL-RGD nerve conduits and PLGL nerve conduits. The effect is as good as that of an autograft. This work established the platform for further development of the use of PLGL-RGD-NGF nerve conduits for clinical nerve repair.
doi:10.1186/1475-925X-11-36
PMCID: PMC3465232  PMID: 22776032
RGD peptide; Nerve growth factor; Peripheral nerve; Nerve conduits; Nerve regeneration
4.  Long-Term Survival and Integration of Transplanted Engineered Nervous Tissue Constructs Promotes Peripheral Nerve Regeneration 
Tissue Engineering. Part A  2009;15(7):1677-1685.
Although peripheral nerve injury is a common consequence of trauma or surgery, there are insufficient means for repair. In particular, there is a critical need for improved methods to facilitate regeneration of axons across major nerve lesions. Here, we engineered transplantable living nervous tissue constructs to provide a labeled pathway to guide host axonal regeneration. These constructs consisted of stretch-grown, longitudinally aligned living axonal tracts inserted into poly(glycolic acid) tubes. The constructs (allogenic) were transplanted to bridge an excised segment of sciatic nerve in the rat, and histological analyses were performed at 6 and 16 weeks posttransplantation to determine graft survival, integration, and host regeneration. At both time points, the transplanted constructs were found to have maintained their pretransplant geometry, with surviving clusters of graft neuronal somata at the extremities of the constructs spanned by tracts of axons. Throughout the transplanted region, there was an intertwining plexus of host and graft axons, suggesting that the transplanted axons mediated host axonal regeneration across the lesion. By 16 weeks posttransplant, extensive myelination of axons was observed throughout the transplant region. Further, graft neurons had extended axons beyond the margins of the transplanted region, penetrating into the host nerve. Notably, this survival and integration of the allogenic constructs occurred in the absence of immunosuppression therapy. These findings demonstrate the promise of living tissue-engineered axonal constructs to bridge major nerve lesions and promote host regeneration, potentially by providing axon-mediated axonal outgrowth and guidance.
doi:10.1089/ten.tea.2008.0294
PMCID: PMC2792099  PMID: 19231968
5.  Repair of Peripheral Nerve Defects Using a Polyvinylidene Fluoride Channel Containing Nerve Growth Factor and Collagen Gel in Adult Rats 
Cell Journal (Yakhteh)  2011;13(3):137-142.
Objective:
As effectiveness of the autologous graft in the repair of long nerve defects is very limited an effective substitute is needed. This study was conducted to determine the poled polyvinylidene fluoride (PVDF) tube as an alternative to nerve autograft.
Materials and Methods:
The left sciatic nerve was transected in 45 male Wistar rats. The animals were then divided randomly into three groups: in an epineural group the nerve was sutured end to end; in an autograft group a 10 mm piece of sciatic nerve was cut, rotated 180° and sutured in the nerve gap; and in a nerve guidance channel group (NGC), PVDF, tube containing nerve growth factor (NGF) and collagen gel was placed in the gap. In a control (n=15) group the sciatic nerve was exposed but not transected. To determine axonal regeneration, retrograde DiI tracer was injected into the gastrocnemius muscle. One week later, retrograde-labeled neurons were counted in the L4-L6 spinal segments and one way ANOVA analysis was performed to compare groups. Neuronal morphology changes were studied by electron microscopy.
Results:
Significant statistical decreases in the mean number of labeled motoneurons were observed in all surgical groups compared to the control group; and in the autograft and the NGC groups compared to epinural suture group (p<0.01). No significant difference in the mean number of motoneurons was observed between the autograft and NGC groups. Chromatin condensation, dilated endoplasmic reticulum and large vacuoles were observed in the autograft and NGC groups.
Conclusion:
Regarding the positive effects of PVDF tube containing NGF and Collagen gel on the sciatic nerve regeneration, authors suggest that it may be useful in peripheral nerve repair.
PMCID: PMC3584471  PMID: 23508804
NGF; Sciatic Nerve; Nerve Injury; Spinal Cord; Motor Neuron
6.  Matching of motor-sensory modality in the rodent femoral nerve model shows no enhanced effect on peripheral nerve regeneration 
Experimental neurology  2010;223(2):496-504.
The treatment of peripheral nerve injuries with nerve gaps largely consists of autologous nerve grafting utilizing sensory nerve donors. Underlying this clinical practice is the assumption that sensory autografts provide a suitable substrate for motoneuron regeneration, thereby facilitating motor endplate reinnervation and functional recovery. This study examined the role of nerve graft modality on axonal regeneration, comparing motor nerve regeneration through motor, sensory, and mixed nerve isografts in the Lewis rat. A total of 100 rats underwent grafting of the motor or sensory branch of the femoral nerve with histomorphometric analysis performed after 5, 6, or 7 weeks. Analysis demonstrated similar nerve regeneration in motor, sensory, and mixed nerve grafts at all three time points. These data indicate that matching of motor-sensory modality in the rat femoral nerve does not confer improved axonal regeneration through nerve isografts.
doi:10.1016/j.expneurol.2010.01.016
PMCID: PMC2865885  PMID: 20122927
Femoral nerve; preferential motor regeneration; nerve architecture; motor graft; sensory graft; modality-specific regeneration
7.  Nerve regeneration over a 25 mm gap in rat sciatic nerves using tubes containing blood vessels: the possibility of clinical application 
International Orthopaedics  1997;21(5):332-336.
Summary.
This study was undertaken to investigate the effect of including vessels in a tube used to promote nerve regeneration across a gap. A tube containing sural vessels was designed in a rat model and interposed between the proximal and distal stumps of a divided sciatic nerve, leaving a 25 mm gap. At 12 weeks, a few myelinated axons were seen at the most distal parts of regenerated nerves in 6 out of 10 rats, none of which evoked action potentials in the tibialis anterior muscle, but by 24 weeks all the rats had developed neural tissue in the tubes, which evoked action potentials in the muscle. The vessels within the tube enhanced nerve regeneration and its distance up 25 mm. This type of vessel-containing tube would be useful for the repair of divided human peripheral nerves with long gaps, almost equivalent to or slightly longer than the maximum length over which nerve fibres can regenerate through a unvascularised unmodified tube.
doi:10.1007/s002640050179
PMCID: PMC3617795  PMID: 9476165
8.  The role of aligned polymer fiber-based constructs in the bridging long peripheral nerve gaps 
Biomaterials  2008;29(21):3117-3127.
Peripheral nerve regeneration across long nerve gaps is clinically challenging. Autografts, the standard of therapy, are limited by availability and other complications. Here, using rigorous anatomical and functional measures, we report that aligned polymer fiber-based constructs present topographical cues that facilitate the regeneration of peripheral nerves across long nerve gaps. Significantly, aligned but not randomly oriented fibers elicit regeneration, establishing that topographical cues can influence endogenous nerve repair mechanisms in the absence of exogenous growth promoting proteins. Axons regenerated across a 17mm nerve gap, reinnervated muscles, and reformed neuromuscular junctions. Electrophysiological and behavioral analyses revealed that aligned, but not randomly oriented constructs facilitated both sensory and motor nerve regeneration, significantly improved functional outcomes. Additionally, a quantitative comparison of DRG outgrowth in vitro and nerve regeneration in vivo on aligned and randomly oriented fiber films clearly demonstrated the significant role of sub-micron scale topographical cues in stimulating endogenous nerve repair mechanisms.
doi:10.1016/j.biomaterials.2008.03.042
PMCID: PMC2483242  PMID: 18448163
9.  Chondroitinase Applied to Peripheral Nerve Repair Averts Retrograde Axonal Regeneration 
Experimental neurology  2006;203(1):185-195.
Antegrade, target-directed axonal regeneration is the explicit goal of nerve repair. However, aberrant and dysfunctional regrowth is commonly observed as well. At the site of surgical nerve coaptation axonal sprouts encounter fibrotic connective tissue rich in growth-inhibiting chondroitin sulfate proteoglycan that may contribute to misdirection of axonal regrowth. In the present study we tested the hypothesis that degradation of chondroitin sulfate proteoglycan by application of chondroitinase at the site of nerve repair can decrease aberrant axonal growth. Adult rats received bilateral sciatic nerve transection and end-to-end repair. One nerve was injected with chondroitinase ABC and the contralateral nerve treated with vehicle alone. After 28 weeks, retrograde axonal regeneration was assessed proximal to the repair by scoring neurofilament-immunopositive axons within the nerve (intrafascicular) and outside the nerve proper (extrafascicular). Intrafascicular retrograde axonal growth was equivalent in both control and chondroitinase treatment conditions. In contrast, chondroitinase treatment caused a pronounced (93%) reduction in extrafascicular retrograde axonal growth. The decrease in axon egress from the nerve was coincident with an increase in antegrade regeneration and improved recovery of motor function. Based on these findings we conclude that chondroitinase applied at the site of nerve transection repair averts dysfunctional extrafascicular retrograde axonal growth.
doi:10.1016/j.expneurol.2006.08.004
PMCID: PMC1851897  PMID: 16970940
chondroitinase; chondroitin sulfate proteoglycan; retrograde regeneration; nerve injury; nerve repair; peripheral nerve; morphometry
10.  The Impact of Motor and Sensory Nerve Architecture on Nerve Regeneration 
Experimental neurology  2008;212(2):370-376.
Sensory nerve autografting is the standard of care for injuries resulting in a nerve gap. Recent work demonstrates superior regeneration with motor nerve grafts. Improved regeneration with motor grafting may be a result of the nerve’s Schwann cell basal lamina tube size. Motor nerves have larger SC basal lamina tubes, which may allow more nerve fibers to cross a nerve graft repair. Architecture may partially explain the suboptimal clinical results seen with sensory nerve grafting techniques. To define the role of nerve architecture, we evaluated regeneration through acellular motor and sensory nerve grafts. Thirty-six Lewis rats underwent tibial nerve repairs with 5 mm double-cable motor or triple-cable sensory nerve isografts. Grafts were harvested and acellularized in University of Wisconsin solution. Control animals received fresh motor or sensory cable isografts. Nerves were harvested after 4 weeks and histomorphometry was performed. In 6 animals per group from the fresh motor and sensory cable graft groups, weekly walking tracks and wet muscle mass ratios were performed at 7 weeks. Histomorphometry revealed more robust nerve regeneration in both acellular and cellular motor grafts. Sensory groups showed poor regeneration with significantly decreased percent nerve, fiber count, and density (p < 0.05). Walking tracks revealed a trend toward improved functional recovery in the motor group. Gastrocnemius wet muscle mass ratios show a significantly greater muscle mass recovery in the motor group (p < 0.05). Nerve architecture (size of SC basal lamina tubes) plays an important role in nerve regeneration in a mixed nerve gap model.
doi:10.1016/j.expneurol.2008.04.012
PMCID: PMC2761727  PMID: 18550053
nerve regeneration; peripheral nerve; motor nerve; preferential motor reinnervation; nerve architecture; acellularized nerve; nerve graft; sensory nerve; nerve transfer
11.  Rolipram-induced elevation of cAMP or chondroitinase ABC breakdown of inhibitory proteoglycans in the extracellular matrix promotes peripheral nerve regeneration 
Experimental neurology  2009;223(1):143-152.
The inhibitory growth environment of myelin and extracellular matrix proteoglycans in the central nervous system may be overcome by elevating neuronal cAMP or degrading inhibitory proteoglycans with chondroitinase ABC (ChABC). In this study, we asked whether similar mechanisms operate in peripheral nerve regeneration where effective Wallerian degeneration removes myelin and extracellular proteoglycans slowly. We repaired transected common peroneal (CP) nerve in rats and either elevated cAMP in the axotomized neurons by subcutaneous rolipram, a specific inhibitor of phosphodiesterase IV, and/or promoted degradation of proteoglycans in the distal nerve stump by local ChABC administration. Rolipram treatment significantly increased the number of motoneurons that regenerated axons across the repair site at one and two weeks, and increased the number of sensory neurons that regenerated axons across the repair site at two weeks. Local application of ChABC had a similar effect to rolipram treatment in promoting motor axon regeneration, the effect being no greater when rolipram and ChABC were administered simultaneously. We conclude that blocking inhibitors of axon regeneration by elevating cAMP or degrading proteoglycans in the distal nerve stump promotes peripheral axon regeneration after surgical repair of a transected nerve. It is likely that elevated cAMP is sufficient to encourage axon outgrowth despite the the inhibitory growth environment such that simultaneous enzymatic proteoglycan degradation does not promote more axon regeneration than either elevated cAMP or proteoglycan degradation alone.
doi:10.1016/j.expneurol.2009.08.026
PMCID: PMC3071985  PMID: 19733561
cAMP; chondroitinase ABC; axon regeneration; axon outgrowth; staggered regeneration; motoneurons; DRG neurons
12.  Sustained Growth Factor Delivery Promotes Axonal Regeneration in Long Gap Peripheral Nerve Repair 
Tissue Engineering. Part A  2011;17(9-10):1263-1275.
The aim of this study was to evaluate the long-term effect of localized growth factor delivery on sciatic nerve regeneration in a critical-size (>1 cm) peripheral nerve defect. Previous work has demonstrated that bioactive proteins can be encapsulated within double-walled, poly(lactic-co-glycolic acid)/poly(lactide) microspheres and embedded within walls of biodegradable polymer nerve guides composed of poly(caprolactone). Within this study, nerve guides containing glial cell line-derived neurotrophic factor (GDNF) were used to bridge a 1.5-cm defect in the male Lewis rat for a 16-week period. Nerve repair was evaluated through functional assessment of joint angle range of motion using video gait kinematics, gastrocnemius twitch force, and gastrocnemius wet weight. Histological evaluation of nerve repair included assessment of Schwann cell and neurofilament location with immunohistochemistry, evaluation of tissue integration and organization throughout the lumen of the regenerated nerve with Masson's trichrome stain, and quantification of axon fiber density and g-ratio. Results from this study showed that the measured gastrocnemius twitch force in animals treated with GDNF was significantly higher than negative controls and was not significantly different from the isograft-positive control group. Histological assessment of explanted conduits after 16 weeks showed improved tissue integration within GDNF releasing nerve guides compared to negative controls. Nerve fibers were present across the entire length of GDNF releasing guides, whereas nerve fibers were not detectable beyond the middle region of negative control guides. Therefore, our results support the use of GDNF for improved functional recovery above negative controls following large axonal defects in the peripheral nervous system.
doi:10.1089/ten.tea.2010.0507
PMCID: PMC3079170  PMID: 21189072
13.  Thin-film enhanced nerve guidance channels for peripheral nerve repair 
Biomaterials  2009;30(23-24):3834-3846.
It has been demonstrated that nerve guidance channels containing stacked thin-films of aligned poly(acrylonitrile-methacrylate) fibers support peripheral nerve regeneration across critical sized nerve gaps, without the aid of exogenous cells or proteins. Here, we explore the ability of tubular channels mininally supplemented with aligned nanofiber-based thin-films to promote endogenous nerve repair. We describe a technique for fabricating guidance channels in which individual thin-films are fixed into place within the lumen of a polysulfone tube. Because each thin-film is <10μm thick, this technique allows fine control over the positioning of aligned scaffolding substrate. We evaluated nerve regeneration through a 1-film guidance channel - containing a single continuous thin-film of aligned fibers - in comparison to a 3-film channel that provided two additional thin-film tracks. Thirty rats were implanted with one of the two channel types, and regeneration across a 14 mm tibial nerve gap was evaluated after 6 weeks and 13 weeks, using a range of morphological and functional measures. Both the 1-film and the 3-film channels supported regeneration across the nerve gap resulting in functional muscular reinnervation. Each channel type characteristically influenced the morphology of the regeneration cable. Interestingly, the 1-film channels supported enhanced regeneration compared to the 3-film channels in terms of regenerated axon profile counts and measures of nerve conduction velocity. These results suggest that minimal levels of appropriately positioned topographical cues significantly enhance guidance channel function by modulating endogenous repair mechanisms, resulting in effective bridging of critically sized peripheral nerve gaps.
doi:10.1016/j.biomaterials.2009.04.022
PMCID: PMC2753861  PMID: 19446873
14.  Immunohistochemical, Ultrastructural and Functional Analysis of Axonal Regeneration through Peripheral Nerve Grafts Containing Schwann Cells Expressing BDNF, CNTF or NT3 
PLoS ONE  2013;8(8):e69987.
We used morphological, immunohistochemical and functional assessments to determine the impact of genetically-modified peripheral nerve (PN) grafts on axonal regeneration after injury. Grafts were assembled from acellular nerve sheaths repopulated ex vivo with Schwann cells (SCs) modified to express brain-derived neurotrophic factor (BDNF), a secretable form of ciliary neurotrophic factor (CNTF), or neurotrophin-3 (NT3). Grafts were used to repair unilateral 1 cm defects in rat peroneal nerves and 10 weeks later outcomes were compared to normal nerves and various controls: autografts, acellular grafts and grafts with unmodified SCs. The number of regenerated βIII-Tubulin positive axons was similar in all grafts with the exception of CNTF, which contained the fewest immunostained axons. There were significantly lower fiber counts in acellular, untransduced SC and NT3 groups using a PanNF antibody, suggesting a paucity of large caliber axons. In addition, NT3 grafts contained the greatest number of sensory fibres, identified with either IB4 or CGRP markers. Examination of semi- and ultra-thin sections revealed heterogeneous graft morphologies, particularly in BDNF and NT3 grafts in which the fascicular organization was pronounced. Unmyelinated axons were loosely organized in numerous Remak bundles in NT3 grafts, while the BDNF graft group displayed the lowest ratio of umyelinated to myelinated axons. Gait analysis revealed that stance width was increased in rats with CNTF and NT3 grafts, and step length involving the injured left hindlimb was significantly greater in NT3 grafted rats, suggesting enhanced sensory sensitivity in these animals. In summary, the selective expression of BDNF, CNTF or NT3 by genetically modified SCs had differential effects on PN graft morphology, the number and type of regenerating axons, myelination, and locomotor function.
doi:10.1371/journal.pone.0069987
PMCID: PMC3739754  PMID: 23950907
15.  Guided regeneration with resorbable conduits in experimental peripheral nerve injuries 
International Orthopaedics  2000;24(3):121-125.
Abstract 
Guided tissue regeneration is a new approach in the reconstructive surgery of peripheral nerves. Artificial conduits can be constructed from biodegradable polymers. Lactic/caproic acid copolymers and polyphospazenes are biocompatible materials with a slow resorption rate. Conduits made from either poly-[l-lactide-co-6-caprolatone] or poly-[bis-(ethylalanate)-phosphazene] were assessed for use as guides for nerve regeneration in experimental animals. Under general anesthesia and by using a microsurgery technique both sciatic nerves were exposed in 2 groups of 9 Wistar rats. On the right side, a 10 mm segment of the nerve was removed, and the defect was then repaired using a conduit. On the left side, the same defect was bridged using as an autograft the nerve segment, which had been removed from the right sciatic nerve. Histological and electron microscopy investigations were performed after 30, 90 and 180 days and showed the gradual degradation of both types of conduits without any evidence of local toxicity. The regeneration of the nerve fibers in the lumen was not significantly different from that shown by the autologous grafts. Likewise, no differences were found at 180 days in the functional recovery of the nerve (evoked muscle action potential). Both conduits were found to be effective for guided nerve regeneration. Poly-[l-lactide-co-6-caprolactone] tubes were easier to insert, while polyphosphazene conduits allowed the use of neurite-promoting factors.
doi:10.1007/s002640000142
PMCID: PMC3619877  PMID: 10990379
16.  Enhanced peripheral nerve regeneration by the combination of a polycaprolactone tubular prosthesis and a scaffold of collagen with supramolecular organization 
Brain and Behavior  2013;3(4):417-430.
The purpose of this study was to investigate the influence of implanting collagen with a supramolecular organization on peripheral nerve regeneration, using the sciatic nerve tubulization technique. For this purpose, adult female Sprague Dawley rats were divided into five groups: (1) TP – sciatic nerve repaired with empty polyethylene tubular prothesis (n = 10), (2) TPCL – nerve repair with empty polycaprolactone (PCL) tubing (n = 8), (3) TPCLF – repair with PCL tubing filled with an implant of collagen with a supramolecular organization (n = 10), (4) AG – animals that received a peripheral nerve autograft (n = 8), and (5) Normal nerves (n = 8). The results were assessed by quantification of the regenerated fibers, nerve morphometry, and transmission electron microscopy, 60 days after surgery. Immunohistochemistry and polarization microscopy were also used to analyze the regenerated nerve structure and cellular elements. The results showed that the AG group presented a larger number of regenerated axons. However, the TPCL and TPCLF groups presented more compact regenerated fibers with a morphometric profile closer to normal, both at the tube midpoint and 2 mm distal to the prosthesis. These findings were reinforced by polarization microscopy, which indicated a better collagen/axons suprastructural organization in the TPCLF derived samples. In addition, the immunohistochemical results obtained using the antibody anti-p75NTR as a Schwann cell reactivity marker demonstrated that the Schwann cells were more reactive during the regenerative process in the TPCLF group as compared to the TPCL group and the normal sciatic nerve. Altogether, the results of this study indicated that the implant of collagen with a supramolecular organization positively influenced and stimulated the regeneration process through the nerve gap, resulting in the formation of a better morphologically arranged tissue.
doi:10.1002/brb3.145
PMCID: PMC3869682  PMID: 24381812
Biomaterials; collagen; nerve regeneration; polarization microscopy; tubulization
17.  Effect of Delayed Peripheral Nerve Repair on Nerve Regeneration, Schwann Cell Function and Target Muscle Recovery 
PLoS ONE  2013;8(2):e56484.
Despite advances in surgical techniques for peripheral nerve repair, functional restitution remains incomplete. The timing of surgery is one factor influencing the extent of recovery but it is not yet clearly defined how long a delay may be tolerated before repair becomes futile. In this study, rats underwent sciatic nerve transection before immediate (0) or 1, 3, or 6 months delayed repair with a nerve graft. Regeneration of spinal motoneurons, 13 weeks after nerve repair, was assessed using retrograde labeling. Nerve tissue was also collected from the proximal and distal stumps and from the nerve graft, together with the medial gastrocnemius (MG) muscles. A dramatic decline in the number of regenerating motoneurons and myelinated axons in the distal nerve stump was observed in the 3- and 6-months delayed groups. After 3 months delay, the axonal number in the proximal stump increased 2–3 folds, accompanied by a smaller axonal area. RT-PCR of distal nerve segments revealed a decline in Schwann cells (SC) markers, most notably in the 3 and 6 month delayed repair samples. There was also a progressive increase in fibrosis and proteoglycan scar markers in the distal nerve with increased delayed repair time. The yield of SC isolated from the distal nerve segments progressively fell with increased delay in repair time but cultured SC from all groups proliferated at similar rates. MG muscle at 3- and 6-months delay repair showed a significant decline in weight (61% and 27% compared with contra-lateral side). Muscle fiber atrophy and changes to neuromuscular junctions were observed with increased delayed repair time suggestive of progressively impaired reinnervation. This study demonstrates that one of the main limiting factors for nerve regeneration after delayed repair is the distal stump. The critical time point after which the outcome of regeneration becomes too poor appears to be 3-months.
doi:10.1371/journal.pone.0056484
PMCID: PMC3567071  PMID: 23409189
18.  Allotransplanted Neurons Used to Repair Peripheral Nerve Injury Do Not Elicit Overt Immunogenicity 
PLoS ONE  2012;7(2):e31675.
A major problem hindering the development of autograft alternatives for repairing peripheral nerve injuries is immunogenicity. We have previously shown successful regeneration in transected rat sciatic nerves using conduits filled with allogeneic dorsal root ganglion (DRG) cells without any immunosuppression. In this study, we re-examined the immunogenicity of our DRG neuron implanted conduits as a potential strategy to overcome transplant rejection. A biodegradable NeuraGen® tube was infused with pure DRG neurons or Schwann cells cultured from a rat strain differing from the host rats and used to repair 8 mm gaps in the sciatic nerve. We observed enhanced regeneration with allogeneic cells compared to empty conduits 16 weeks post-surgery, but morphological analyses suggest recovery comparable to the healthy nerves was not achieved. The degree of regeneration was indistinguishable between DRG and Schwann cell allografts although immunogenicity assessments revealed substantially increased presence of Interferon gamma (IFN-γ) in Schwann cell allografts compared to the DRG allografts by two weeks post-surgery. Macrophage infiltration of the regenerated nerve graft in the DRG group 16 weeks post-surgery was below the level of the empty conduit (0.56 fold change from NG; p<0.05) while the Schwann cell group revealed significantly higher counts (1.29 fold change from NG; p<0.001). Major histocompatibility complex I (MHC I) molecules were present in significantly increased levels in the DRG and Schwann cell allograft groups compared to the hollow NG conduit and the Sham healthy nerve. Our results confirmed previous studies that have reported Schwann cells as being immunogenic, likely due to MHC I expression. Nerve gap injuries are difficult to repair; our data suggest that DRG neurons are superior medium to implant inside conduit tubes due to reduced immunogenicity and represent a potential treatment strategy that could be preferable to the current gold standard of autologous nerve transplant.
doi:10.1371/journal.pone.0031675
PMCID: PMC3276507  PMID: 22347502
19.  Treadmill Training Enhances Axon Regeneration In Injured Mouse Peripheral Nerves Without Increased Loss of Topographic Specificity 
We investigated the extent of misdirection of regenerating axons when that regeneration was enhanced using treadmill training. Retrograde fluorescent tracers were applied to the cut proximal stumps of the tibial and common fibular nerves two or four weeks after transection and surgical repair of the mouse sciatic nerve. The spatial locations of retrogradely labeled motoneurons were studied in untreated control mice and in mice receiving two weeks of treadmill training, either according to a continuous protocol (10 m/min, one hour/day, five day/week) or an interval protocol (20 m/min for two minutes, followed by a five minute rest, repeated 4 times, five days/week). More retrogradely labeled motoneurons were found in both treadmill trained groups. The magnitude of this increase was as great as or greater than that found after using other enhancement strategies. In both treadmill trained groups, the proportions of motoneurons labeled from tracer applied to the common fibular nerve that were found in spinal cord locations reserved for tibial motoneurons in intact mice was no greater than in untreated control mice and significantly less than found after electrical stimulation or chondroitinase treatment. Treadmill training in the first two weeks following peripheral nerve injury produces a marked enhancement of motor axon regeneration without increasing the propensity of those axons to choose pathways leading to functionally inappropriate targets.
doi:10.1002/cne.22149
PMCID: PMC2804895  PMID: 19731339
exercise; motoneurons; retrograde tracing; spinal cord
20.  Ultrastructural Changes in Spinal Motoneurons and Locomotor Functional Study after Sciatic Nerve Repair in Conduit Tube 
Objective(s)
Motor deficit and neuron degeneration is seen after nerve transection. The aim of this study is to determine whether a poled polyvinelidene fluoride (PVDF) tube with other supportive strategies can protect the neuronal morphology and motor function after sciatic nerve transaction in rats.
Materials and Methods
After transection of the left sciatic nerve in 60 male Wistar rats (200-250 g), the epineural group was sutured end to end. In the autograft rats, a 10 mm piece of sciatic nerve was rotated 180 °C and sutured back into the nerve gap. In the nerve guidance channel (NGC) group, polarized piezoelectric PVDF tube containing NGF and collagen gel was sutured in the gap. In control group sciatic nerve was removed (10 mm) without repair. After one, four and eight weeks, the L4-L6 spinal cord segment was removed for histological study using transmission electron microscope. Functional outcome was assessed using the Basso, Bresnahan and Beattie (BBB) locomotor scale at both four and eight weeks after the lesion.
Results
Chromatin condensation was seen after 4 weeks in the repair groups. Cell membrane shrinkage and mitochondrial degeneration was observed after 4 and 8 weeks respectively, in the autografted and NGC rats. In the control group, chromatin condensation, cell membrane shrinkage with mitochondrial degeneration and vacuolization of perikaryon was seen after 1, 4 and 8 weeks, respectively. At 56 days, the functional recovery of the epineural rats significantly increased in comparison to the other groups (P< 0.05).
Conclusion
The epineural suture has more efficacies, and NGC may be used as a proper substitute for autograft in nerve injury.
PMCID: PMC3586910  PMID: 23492837
Motor neuron; Rat; Recovery; Sciatic nerve
21.  The relationship between nerve conduction velocity and fiber morphology during peripheral nerve regeneration 
Brain and Behavior  2012;2(4):382-390.
We analyzed the relationship between motor nerve conduction velocity (MCV) and morphological changes in regenerating nerve fibers at different times after sciatic nerve transection to identify reliable indices of functional recovery. Thirty rats were divided into five equal groups, one control group and four groups subjected to sciatic nerve transection and immediate suturing, followed by regeneration for 50, 100, 150, and 200 days, respectively. MCV was measured in each group, followed by morphometric analyses of fibers of the common peroneal nerve. MCV increased progressively with time after nerve transection, although it remained lower than the control velocity. Mean fiber diameter (axon plus myelin sheath) also increased with time after nerve transection. Recovery of mean fiber diameter was well correlated with MCV, even though regenerating nerves likely contained many small nonconducting fibers. In contrast, the change in the mean diameter of regenerating axons and relative myelin thickness (g-ratio) did not provide an accurate measure of recovery as they were not increasing in a time-dependent manner. Furthermore, internodal length changed only slightly with increasing fiber diameter in regenerating nerves; therefore, the regression relation between fiber diameter and internodal length was not a sensitive index of recovery. MCV and mean fiber diameter were the most sensitive indices of functional recovery during sciatic nerve regeneration.
doi:10.1002/brb3.61
PMCID: PMC3432961  PMID: 22950042
Axon diameter; fiber diameter; g-ratio; internodal length; motor nerve conduction velocity; nerve regeneration
22.  Selectivity in the Reinnervation of the Lateral Gastrocnemius Muscle after Nerve Repair with Ethyl Cyanoacrylate in the Rat 
There is a need for complementary surgical techniques that enable rapid and reliable primary repair of transected nerves. Previous studies after peripheral nerve transection and repair with synthetic adhesives have demonstrated regeneration to an extent comparable to that of conventional techniques. The aim of this study was to compare two different repair techniques on the selectivity of muscle reinnervation after repair and completed regeneration. We used the cholera toxin B technique of retrograde axonal tracing to evaluate the morphology, the number, and the three-dimensional location of α-motoneurons innervating the lateral gastrocnemius muscle and compared the results after repair with either ethyl cyanoacrylate (ECA) or epineural sutures of the transected parent sciatic nerve. In addition, we recorded the wet weight of the muscle. Six months after transection and repair of the sciatic nerve, the redistribution of the motoneuron pool was markedly disorganized, the motoneurons had apparently increased in number, and they were scattered throughout a larger volume of the spinal cord gray matter with a decrease in the synaptic coverage compared to controls. A reduction in muscle weight was observed as well. No difference in morphometric variables or muscle weight between the two repair methods could be detected. We conclude that the selectivity of motor reinnervation following sciatic nerve transection and subsequent repair with ECA is comparable to that following conventional micro suturing.
doi:10.3389/fneur.2011.00025
PMCID: PMC3087165  PMID: 21577248
nerve repair; cyanoacrylate; synthetic adhesive; retrograde tracing; gastrocnemius muscle; spinal misdirection; sciatic nerve; peripheral nerve
23.  Sensory axon targeting is increased by NGF gene therapy within the lesioned adult femoral nerve 
Experimental neurology  2009;223(1):153-165.
Even though peripheral nerves regenerate well, axons are often misrouted and reinnervate inappropriate distal pathways post-injury. Misrouting most likely occurs at branch points where regenerating axons make choices. Here, we show that the accuracy of sensory axon reinnervation is enhanced by overexpression of the guidance molecule nerve growth factor (NGF) distal to the bifurcation. We used the femoral nerve as model, which contains both sensory and motor axons that intermingle in the parent trunk and distally segregate into the saphenous (SB) and motor branch (MB). Transection of the parent trunk resulted in misrouting of axon reinnervation to SB and MB. To enhance sensory axon targeting, recombinant adenovirus encoding NGF was injected along the SB close to the bifurcation one week post-injury. The accuracy of axon reinnervation was assessed by retrograde tracing at 3 or 8 weeks after nerve injury. NGF overexpression significantly increased the accuracy of SB axon reinnervation to the appropriate nerve branch, in a manner independent of enhancing axon regeneration. This novel finding provides in vivo evidence that gradient expression of neurotrophin can be used to enhance targeting of distal peripheral pathways to increase axon regeneration into the appropriate nerve branch.
doi:10.1016/j.expneurol.2009.08.025
PMCID: PMC2849876  PMID: 19733564
femoral transection; axon reinnervation; recombinant adenovirus; nerve growth factor; axon guidance; axon targeting; triple retrograde tracing
24.  Using Eggshell Membrane as Nerve Guide Channels in Peripheral Nerve Regeneration 
Objective(s): The aim of this study was to evaluate the final outcome of nerve regeneration across the eggsell membrane (ESM) tube conduit in comparison with autograft.
Materials and Methods: Thirty adult male rats (250-300 g) were randomized into (1) ESM conduit, (2) autograft, and (3) sham surgery groups. The eggs submerged in 5% acetic acid. The decalcifying membranes were cut into four pieces, rotated over the teflon mandrel and dried at 37°C. The left sciatic nerve was surgically cut. A 10-mm nerve segment was cut and removed. In the ESM group, the proximal and distal cut ends of the sciatic nerve were telescoped into the nerve guides. In the autograft group, the 10 mm nerve segment was reversed and used as an autologous nerve graft. All animals were evaluated by sciatic functional index (SFI) and electrophysiology testing.
Results: The improvement in SFI from the first to the last evalution in ESM and autograft groups were evaluated. On days 49 and 60 post-operation, the mean SFI of ESM group was significantly greater than the autograft group (P< 0.05). On day 90, the mean nerve conduction velocity (NCV) of ESM group was greater than autograft group, although the difference was not statistically significant (P> 0.05).
Conclusion: These findings demonstrate that ESM effectively enhances nerve regeneration and promotes functional recovery in injured sciatic nerve of rat.
PMCID: PMC3786101  PMID: 24106593
Eggshell membrane; Nerve regeneration; Rat; Sciatic nerve
25.  The Impact of Motor Axon Misdirection and Attrition on Behavioral Deficit Following Experimental Nerve Injuries 
PLoS ONE  2013;8(11):e82546.
Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (p<.0001, r2=.67), motor neuron counts (attrition) (p<.0001, r2=.69) and final functional deficits. We demonstrate prominent motor axon misdirection and attrition in neuroma-in-continuity and transection injuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly demonstrate these correlations with data inclusive of the neuroma-in-continuity spectrum. This work emphasizes the need to focus on strategies that promote both robust and accurate nerve regeneration to optimize functional recovery. It also demonstrates that clinically relevant neuroma-in-continuity injuries can now also be subjected to experimental investigation.
doi:10.1371/journal.pone.0082546
PMCID: PMC3839879  PMID: 24282624

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