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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.  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
3.  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
4.  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
5.  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
6.  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
7.  A Novel Internal Fixator Device for Peripheral Nerve Regeneration 
Recovery from peripheral nerve damage, especially for a transected nerve, is rarely complete, resulting in impaired motor function, sensory loss, and chronic pain with inappropriate autonomic responses that seriously impair quality of life. In consequence, strategies for enhancing peripheral nerve repair are of high clinical importance. Tension is a key determinant of neuronal growth and function. In vitro and in vivo experiments have shown that moderate levels of imposed tension (strain) can encourage axonal outgrowth; however, few strategies of peripheral nerve repair emphasize the mechanical environment of the injured nerve. Toward the development of more effective nerve regeneration strategies, we demonstrate the design, fabrication, and implementation of a novel, modular nerve-lengthening device, which allows the imposition of moderate tensile loads in parallel with existing scaffold-based tissue engineering strategies for nerve repair. This concept would enable nerve regeneration in two superposed regimes of nerve extension—traditional extension through axonal outgrowth into a scaffold and extension in intact regions of the proximal nerve, such as that occurring during growth or limb-lengthening. Self-sizing silicone nerve cuffs were fabricated to grip nerve stumps without slippage, and nerves were deformed by actuating a telescoping internal fixator. Poly(lactic co-glycolic) acid (PLGA) constructs mounted on the telescoping rods were apposed to the nerve stumps to guide axonal outgrowth. Neuronal cells were exposed to PLGA using direct contact and extract methods, and they exhibited no signs of cytotoxic effects in terms of cell morphology and viability. We confirmed the feasibility of implanting and actuating our device within a sciatic nerve gap and observed axonal outgrowth following device implantation. The successful fabrication and implementation of our device provides a novel method for examining mechanical influences on nerve regeneration.
doi:10.1089/ten.tec.2012.0021
PMCID: PMC3629849  PMID: 23102114
8.  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
9.  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
10.  Long-term changes in neurotrophic factor expression in distal nerve stump following denervation and reinnervation with motor or sensory nerve 
Journal of neurochemistry  2008;105(4):1244-1252.
Several factors have been proposed to account for poor motor recovery after prolonged denervation, including motor neuron cell death and incomplete or poor regeneration of motor fibers into the muscle. Both may result from failure of the muscle and the distal motor nerve stump to continue expression of neurotrophic factors following delayed muscle reinnervation. This study investigated whether regenerating motor or sensory axons modulate distal nerve neurotrophic factor expression. We found that transected distal tibial nerve up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, down-regulated neuro-trophin-3 and ciliary neurotrophic factor mRNA, and that although these levels returned to normal with regeneration, the chronically denervated distal nerve stump continued to express these neurotrophic factors for at least 6 months following injury. A sensory nerve (the cutaneous saphenous nerve) sutured to distal tibial nerve lowered injury-induced BDNF and GDNF mRNA levels in distal stump, but repair with a mixed nerve (peroneal, containing muscle and cutaneous axons) was more effective. Repair with sensory or mixed nerves did not affect nerve growth factor or neurotrophin-3 expression. Thus, distal nerve contributed to a neurotrophic environment for nerve regeneration for at least 6 months, and sensory nerve repair helped normalize distal nerve neurotrophic factor mRNA expression following denervation. Furthermore, as BDNF and GDNF levels in distal stump increased following denervation and returned to control levels following reinnervation, their levels serve as markers for the status of regeneration by either motor or sensory nerve.
doi:10.1111/j.1471-4159.2008.05224.x
PMCID: PMC3414532  PMID: 18194437 CAMSID: cams2286
mRNA; nerve injury; neurotrophins; rat; sciatic nerve
11.  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
12.  Biological and Electrophysiologic Effects of Poly(3,4-ethylenedioxythiophene) on Regenerating Peripheral Nerve Fibers 
Plastic and reconstructive surgery  2013;132(2):374-385.
Background
Uninjured peripheral nerves in upper-limb amputees represent attractive sites for connectivity with neuroprostheses because their predictable internal topography allows for precise sorting of motor and sensory signals. The inclusion of poly(3,4-ethylenedioxythiophene) reduces impedance and improves charge transfer at the biotic-abiotic interface. This study evaluates the in vivo performance of poly(3,4-ethylenedioxythiophene)–coated interpositional decellularized nerve grafts across a critical nerve conduction gap, and examines the long-term effects of two different poly(3,4-ethylenedioxythiophene) formulations on regenerating peripheral nerve fibers.
Methods
In 48 rats, a 15-mm gap in the common peroneal nerve was repaired using a nerve graft of equivalent length, including (1) decellularized nerve chemically polymerized with poly(3,4-ethylenedioxythiophene) (dry); (2) decellularized nerve electrochemically polymerized with poly(3,4-ethylenedioxythiophene) (wet); (3) intact nerve; (4) autogenous nerve graft; (5) decellularized nerve alone; and (6) unrepaired nerve gap controls. All groups underwent electrophysiologic characterization at 3 months, and nerves were harvested for histomorphometric analysis.
Results
Conduction velocity was significantly faster in the dry poly(3,4-ethylenedioxythiophene) group compared with the sham, decellularized nerve, and wet poly(3,4-ethylenedioxythiophene) groups. Maximum specific force for the dry poly(3,4-ethylenedioxythiophene) group was more similar to sham than were decellularized nerve controls. Evident neural regeneration was demonstrated in both dry and wet poly(3,4-ethylenedioxythiophene) groups by the presence of normal regenerating axons on histologic cross-section.
Conclusions
Both poly(3,4-ethylenedioxythiophene) formulations were compatible with peripheral nerve regeneration at 3 months. This study supports poly(3,4-ethylenedioxythiophene) as a promising adjunct for peripheral nerve interfaces for prosthetic control and other biomedical applications because of its recognized ionic-to-electronic coupling potential.
doi:10.1097/PRS.0b013e3182959f63
PMCID: PMC4206183  PMID: 23897336
13.  “Supercharge Nerve Transfer to Enhance Motor Recovery, a Laboratory Study” 
The Journal of hand surgery  2013;38(3):466-477.
Purpose
To investigate the ability of a supercharge end-to-side (SETS) nerve transfer to augment the effect of regenerating native axons in an incomplete rodent sciatic nerve injury model.
Methods
Fifty-four Lewis rats were randomized to 3 groups. The first group was an incomplete recovery model (IRM) of the tibial nerve complemented with a SETS transfer from the peroneal nerve (SETS-IRM). The IRM consisted of tibial nerve transection and immediate repair using a 10mm fresh tibial isograft to provide some, but incomplete, nerve recovery. The 2 control groups were IRM alone and SETS alone.
Nerve histomorphometry, electron microscopy, retrograde labeling, and muscle force testing were performed.
Results
Histomorphometry of the distal tibial nerve showed significantly increased myelinated axonal counts in the SETS-IRM group compared to the IRM and SETS groups at 5 and 8 weeks. Retrograde labeling at 8 weeks confirmed increased motoneuron counts in the SETS-IRM group. Functional recovery at 8 weeks showed a significant increase in muscle specific force in the SETS-IRM group compared to the IRM group.
Conclusions
A SETS transfer enhanced recovery from an incomplete nerve injury as determined by histomorphometry, motoneuron labeling within the spinal cord, and muscle force measurements.
Clinical Relevance
A SETS distal nerve transfer may be useful in nerve injuries with incomplete regeneration such as proximal Sunderland II or III degree injuries, where long regeneration distance yields prolonged time to muscle reinnervation and suboptimal functional recovery.
doi:10.1016/j.jhsa.2012.12.020
PMCID: PMC3583195  PMID: 23391355
Nerve regeneration; nerve transfer; neurorrhaphy; peripheral nerve; supercharge end-to-side
14.  Ciliary neurotrophic factor promotes motor reinnervation of the musculocutaneous nerve in an experimental model of end-to-side neurorrhaphy 
BMC Neuroscience  2011;12:58.
Background
It is difficult to repair nerve if proximal stump is unavailable or autogenous nerve grafts are insufficient for reconstructing extensive nerve damage. Therefore, alternative methods have been developed, including lateral anastomosis based on axons' ability to send out collateral sprouts into denervated nerve. The different capacity of a sensory or motor axon to send a sprout is controversial and may be controlled by cytokines and/or neurotrophic factors like ciliary neurotrophic factor (CNTF). The aim of the present study was to quantitatively assess collateral sprouts sent out by intact motor and sensory axons in the end-to-side neurorrhaphy model following intrathecal administration of CNTF in comparison with phosphate buffered saline (vehiculum) and Cerebrolysin.
The distal stump of rat transected musculocutaneous nerve (MCN) was attached in an end-to-side fashion with ulnar nerve. CNTF, Cerebrolysin and vehiculum were administered intrathecally for 2 weeks, and all animals were allowed to survive for 2 months from operation. Numbers of spinal motor and dorsal root ganglia neurons were estimated following their retrograde labeling by Fluoro-Ruby and Fluoro-Emerald applied to ulnar and musculocutaneous nerve, respectively. Reinnervation of biceps brachii muscles was assessed by electromyography, behavioral test, and diameter and myelin sheath thickness of regenerated axons.
Results
Vehiculum or Cerebrolysin administration resulted in significantly higher numbers of myelinated axons regenerated into the MCN stumps compared with CNTF treatment. By contrast, the mean diameter of the myelinated axons and their myelin sheath thickness in the cases of Cerebrolysin- or CNTF-treated animals were larger than were those for rats treated with vehiculum. CNTF treatment significantly increased the percentage of motoneurons contributing to reinnervation of the MCN stumps (to 17.1%) when compared with vehiculum or Cerebrolysin treatments (at 9.9 or 9.6%, respectively). Reduced numbers of myelinated axons and simultaneously increased numbers of motoneurons contributing to reinnervation of the MCN improved functional reinnervation of the biceps brachii muscle after CNTF treatment.
Conclusion
The present experimental study confirms end-to-side neurorrhaphy as an alternative method for reconstructing severed peripheral nerves. CNTF promotes motor reinnervation of the MCN stump after its end-to-side neurorrhaphy with ulnar nerve and improves functional recovery of the biceps brachii muscle.
doi:10.1186/1471-2202-12-58
PMCID: PMC3224149  PMID: 21696588
15.  Poly(lactic-co-glycolic acid) conduit for repair of injured sciatic nerve: A mechanical analysis 
Neural Regeneration Research  2013;8(21):1966-1973.
Tensile stress and tensile strain directly affect the quality of nerve regeneration after bridging nerve defects by poly(lactic-co-glycolic acid) conduit transplantation and autogenous nerve grafting for sciatic nerve injury. This study collected the sciatic nerve from the gluteus maximus muscle from fresh human cadaver, and established 10-mm-long sciatic nerve injury models by removing the ischium, following which poly(lactic-co-glycolic acid) conduits or autogenous nerve grafts were transplanted. Scanning electron microscopy revealed that the axon and myelin sheath were torn, and the vessels of basilar membrane were obstructed in the poly(lactic-co-glycolic acid) conduit-repaired sciatic nerve following tensile testing. There were no significant differences in tensile tests with autogenous nerve graft-repaired sciatic nerve. Following poly(lactic-co-glycolic acid) conduit transplantation for sciatic nerve repair, tensile test results suggest that maximum tensile load, maximum stress, elastic limit load and elastic limit stress increased compared with autogenous nerve grafts, but elastic limit strain and maximum strain decreased. Moreover, the tendencies of stress-strain curves of sciatic nerves were similar after transplantation of poly(lactic-co-glycolic acid) conduits or autogenous nerve grafts. Results showed that after transplantation in vitro for sciatic nerve injury, poly(lactic-co-glycolic acid) conduits exhibited good intensity, elasticity and plasticity, indicating that poly(lactic-co-glycolic acid) conduits are suitable for sciatic nerve injury repair.
doi:10.3969/j.issn.1673-5374.2013.21.005
PMCID: PMC4145904  PMID: 25206505
neural regeneration; peripheral nerve injury; sciatic nerve; injury model; poly(lactic-co-glycolic acid); transplantation; stress; strain; mechanical property; grants-supported paper; neuroregeneration
16.  Chondroitinase treatment increases the effective length of acellular nerve grafts 
Experimental neurology  2007;207(1):163-170.
Acellular nerve allografts have been explored as an alternative to nerve autografting. It has long been recognized that there is a distinct limit to the effective length of conventional acellular nerve grafts, which must be overcome for many grafting applications. In rodent models nerve regeneration fails in acellular nerve grafts greater than two cm in length. In previous studies we found that nerve regeneration is markedly enhanced with acellular nerve grafts in which growth-inhibiting chondroitin sulfate proteoglycan was degraded by pretreatment with chondroitinase ABC (ChABC). Here, we tested if nerve regeneration can be achieved through 4-cm acellular nerve grafts pretreated with ChABC. Adult rats received bilateral sciatic nerve segmental resection and repair with a four-cm, thermally acellularized, nerve graft treated with ChABC (ChABC graft) or vehicle-treated acellularized graft (Control graft). Nerve regeneration was examined 12 weeks after implantation. Our findings confirm that functional axonal regeneration fails in conventional long acellular grafts. In this condition we found very few axons in the distal host nerve, and there were marginal signs of sciatic nerve reinnervation in few (2/9) rats. This was accompanied by extensive structural disintegration of the distal graft and abundant retrograde axonal regeneration in the proximal nerve. In contrast, most (8/9) animals receiving nerve repair with ChABC grafts showed sciatic nerve reinnervation by direct nerve pinch testing. Histological examination revealed much better structural preservation and axonal growth throughout the ChABC grafts. Numerous axons were found in all but one (8/9) of the host distal nerves and many of these regenerated axons were myelinated. In addition, the amount of aberrant retrograde axonal growth (originating near the proximal suture line) was markedly reduced by repair with ChABC grafts. Based on these results we conclude that ChABC treatment substantially increases the effective length of acellular nerve grafts.
doi:10.1016/j.expneurol.2007.06.006
PMCID: PMC2956445  PMID: 17669401
17.  A simple model of radial nerve injury in the rhesus monkey to evaluate peripheral nerve repair 
Neural Regeneration Research  2014;9(10):1041-1046.
Current research on bone marrow stem cell transplantation and autologous or xenogenic nerve transplantation for peripheral nerve regeneration has mainly focused on the repair of peripheral nerve defects in rodents. In this study, we established a standardized experimental model of radial nerve defects in primates and evaluated the effect of repair on peripheral nerve injury. We repaired 2.5-cm lesions in the radial nerve of rhesus monkeys by transplantation of autografts, acellular allografts, or acellular allografts seeded with autologous bone marrow stem cells. Five months after surgery, regenerated nerve tissue was assessed for function, electrophysiology, and histomorphometry. Postoperative functional recovery was evaluated by the wrist-extension test. Compared with the simple autografts, the acellular allografts and allografts seeded with bone marrow stem cells facilitated remarkable recovery of the wrist-extension functions in the rhesus monkeys. This functional improvement was coupled with radial nerve distal axon growth, a higher percentage of neuron survival, increased nerve fiber density and diameter, increased myelin sheath thickness, and increased nerve conduction velocities and peak amplitudes of compound motor action potentials. Furthermore, the quality of nerve regeneration in the bone marrow stem cells-laden allografts group was comparable to that achieved with autografts. The wrist-extension test is a simple behavioral method for objective quantification of peripheral nerve regeneration.
doi:10.4103/1673-5374.133166
PMCID: PMC4146303  PMID: 25206757
nerve regeneration; peripheral nerve injury; rhesus monkeys; bone marrow stem cells; allogeneic nerve; transplantation; wrist-extension test; electrophysiology; neurological function; NSFC grant; neural regeneration
18.  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
19.  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
20.  Cholecalciferol (Vitamin D3) Improves Myelination and Recovery after Nerve Injury 
PLoS ONE  2013;8(5):e65034.
Previously, we demonstrated i) that ergocalciferol (vitamin D2) increases axon diameter and potentiates nerve regeneration in a rat model of transected peripheral nerve and ii) that cholecalciferol (vitamin D3) improves breathing and hyper-reflexia in a rat model of paraplegia. However, before bringing this molecule to the clinic, it was of prime importance i) to assess which form – ergocalciferol versus cholecalciferol – and which dose were the most efficient and ii) to identify the molecular pathways activated by this pleiotropic molecule. The rat left peroneal nerve was cut out on a length of 10 mm and autografted in an inverted position. Animals were treated with either cholecalciferol or ergocalciferol, at the dose of 100 or 500 IU/kg/day, or excipient (Vehicle), and compared to unlesioned rats (Control). Functional recovery of hindlimb was measured weekly, during 12 weeks, using the peroneal functional index. Ventilatory, motor and sensitive responses of the regenerated axons were recorded and histological analysis was performed. In parallel, to identify the genes regulated by vitamin D in dorsal root ganglia and/or Schwann cells, we performed an in vitro transcriptome study. We observed that cholecalciferol is more efficient than ergocalciferol and, when delivered at a high dose (500 IU/kg/day), cholecalciferol induces a significant locomotor and electrophysiological recovery. We also demonstrated that cholecalciferol increases i) the number of preserved or newly formed axons in the proximal end, ii) the mean axon diameter in the distal end, and iii) neurite myelination in both distal and proximal ends. Finally, we found a modified expression of several genes involved in axogenesis and myelination, after 24 hours of vitamin supplementation. Our study is the first to demonstrate that vitamin D acts on myelination via the activation of several myelin-associated genes. It paves the way for future randomised controlled clinical trials for peripheral nerve or spinal cord repair.
doi:10.1371/journal.pone.0065034
PMCID: PMC3669361  PMID: 23741446
21.  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
22.  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
23.  Effect of Modified Formula Radix Hedysari on the Amplification Effect during Peripheral Nerve Regeneration 
Many studies have demonstrated a compensatory amplification phenomenon during nerve regeneration. When a relatively fine nerve is used as a donor to connect to a distal nerve after transection, the donor nerve regenerates more collaterals than its own fibers, which extend to the distal stump, grow into distal endoneurial tubes, and finally reach and dominate the target organs. This is known as the amplification phenomenon. In this study, we investigated the amplification phenomenon in rats treated with Modified Formula Radix Hedysari (MFRH) as adjuvant therapy for 12 weeks. The rats were divided into three groups at random (six animals in each group). In the model group and the treatment group, the proximal common peroneal nerve was used as a donor nerve to connect to the distal tibial nerve. Rats in the normal group did not undergo surgery. After surgery, the treatment group was administered MFRH as systemic therapy, while the model group and the normal group were not given treatment. The results demonstrated that the nerve conduction velocity, the fiber diameter, the axon diameter, the number of regenerating nerve fibers, and the amplification ratio were better in the treatment group than in the model group, suggesting that MFRH promoted the nerve amplification effect.
doi:10.1155/2013/647982
PMCID: PMC3595679  PMID: 23533510
24.  Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse 
BMC Neuroscience  2007;8:80.
Background
The chondroitin sulphate proteoglycan NG2 blocks neurite outgrowth in vitro and has been proposed as a major inhibitor of axonal regeneration in the CNS. Although a substantial body of evidence underpins this hypothesis, it is challenged by recent findings including strong expression of NG2 in regenerating peripheral nerve.
Results
We studied axonal regeneration in the PNS and CNS of genetically engineered mice that do not express NG2, and in sex and age matched wild-type controls. In the CNS, we used anterograde tracing with BDA to study corticospinal tract (CST) axons after spinal cord injury and transganglionic labelling with CT-HRP to trace ascending sensory dorsal column (DC) axons after DC lesions and a conditioning lesion of the sciatic nerve. Injury to these fibre tracts resulted in no difference between knockout and wild-type mice in the ability of CST axons or DC axons to enter or cross the lesion site. Similarly, after dorsal root injury (with conditioning lesion), most regenerating dorsal root axons failed to grow across the dorsal root entry zone in both transgenic and wild-type mice.
Following sciatic nerve injuries, functional recovery was assessed by analysis of the toe-spreading reflex and cutaneous sensitivity to Von Frey hairs. Anatomical correlates of regeneration were assessed by: retrograde labelling of regenerating dorsal root ganglion (DRG) cells with DiAsp; immunostaining with PGP 9.5 to visualise sensory reinnervation of plantar hindpaws; electron microscopic analysis of regenerating axons in tibial and digital nerves; and by silver-cholinesterase histochemical study of motor end plate reinnervation. We also examined functional and anatomical correlates of regeneration after injury of the facial nerve by assessing the time taken for whisker movements and corneal reflexes to recover and by retrograde labelling of regenerated axons with Fluorogold and DiAsp. None of the anatomical or functional analyses revealed significant differences between wild-type and knockout mice.
Conclusion
These findings show that NG2 is unlikely to be a major inhibitor of axonal regeneration after injury to the CNS, and, further, that NG2 is unlikely to be necessary for regeneration or functional recovery following peripheral nerve injury.
doi:10.1186/1471-2202-8-80
PMCID: PMC2100060  PMID: 17900358
25.  Omentum-Wrapped Scaffold with Longitudinally Oriented Micro-Channels Promotes Axonal Regeneration and Motor Functional Recovery in Rats 
PLoS ONE  2011;6(12):e29184.
Background
Tissue-engineered nerve scaffolds hold great potential in bridging large peripheral nerve defects. However, insufficient vascularization of nerve scaffolds limited neural tissues survival and regeneration, which hampered the successful implantation and clinical application of nerve scaffolds. The omentum possesses a high vascularization capacity and enhances regeneration and maturation of tissues and constructs to which it is applied. However, combined application of nerve scaffolds and omentum on axonal regeneration and functional recovery in the treatment of large peripheral nerve defects has rarely been investigated thus far.
Methods
In the present study, an omentum-wrapped collagen-chitosan scaffold was used to bridge a 15-mm-long sciatic nerve defect in rats. Rats that received nerve autografts or scaffolds alone were served as positive control or negative control, respectively. The axonal regeneration and functional recovery were examined by a combination of walking track analysis, electrophysiological assessment, Fluoro-Gold (FG) retrograde tracing, as well as morphometric analyses to both regenerated nerves and target muscles.
Findings
The results demonstrated that axonal regeneration and functional recovery were in the similar range between the omentum-wrapping group and the autograft group, which were significantly better than those in the scaffold alone group. Further investigation showed that the protein levels of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were significantly higher in the omentum-wrapping group than those in the scaffold alone group in the early weeks after surgery.
Conclusion
These findings indicate that the omentum-wrapped scaffold is capable of enhancing axonal regeneration and functional recovery, which might be served as a potent alternative to nerve autografts. The beneficial effect of omentum-wrapping on nerve regeneration might be related with the proteins produced by omentum.
doi:10.1371/journal.pone.0029184
PMCID: PMC3241706  PMID: 22195018

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