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1.  Beneficial effects of treadmill training in experimental diabetic nerve regeneration 
Clinics  2010;65(12):1329-1337.
We investigated the effects of treadmill training (10 weeks) on hindlimb motor function and nerve morphometric parameters in diabetic rats submitted to sciatic nerve crush.
Wistar rats (n = 64) were divided into the following groups: non-diabetic; trained non-diabetic; non-diabetic with sciatic nerve crush; trained non-diabetic with sciatic nerve crush; diabetic; trained diabetic; diabetic with sciatic nerve crush or trained diabetic with sciatic nerve crush. Diabetes was induced by streptozotocin injection (50 mg/kg, iv). Hindlimb motor function was evaluated weekly by assessing sciatic functional indices, and the proximal and distal portions of the sciatic nerve were used for morphometric analysis.
At 13 weeks post-injury, the distal nerve portion of all injured groups and the proximal nerve portion of the diabetic with sciatic nerve crush group presented altered morphometric parameters such as decreased myelinated fiber diameter (∼7.4±0.3µm vs ∼4.8±0.2µm), axonal diameter (∼5±0.2µm vs ∼3.5±0.1µm) and myelin sheath thickness (∼1.2±0.07µm vs ∼0.65±0.07µm) and an increase in the percentage of area occupied by endoneurium (∼28±3% vs ∼60±3%). In addition, in the non-diabetic with sciatic nerve crush group the proximal nerve portion showed a decreased myelinated fiber diameter (7.4±0.3µm vs 5.8±0.3µm) and myelin sheath thickness (1.29±0.08µm vs 0.92±0.08µm). The non-diabetic with sciatic nerve crush, trained non-diabetic with sciatic nerve crush, diabetic with sciatic nerve crush and trained diabetic with sciatic nerve crush groups showed normal sciatic functional index from the 4th, 4th, 9th and 7th week post-injury, respectively. Morphometric alterations in the proximal nerve portion of the diabetic with sciatic nerve crush and non-diabetic with sciatic nerve crush groups were either prevented or reverted to values similar to the non-diabetic group by treadmill training.
Diabetic condition promoted delay in sciatic nerve regeneration. Treadmill training is able to accelerate hindlimb motor function recovery in diabetic injured rats and prevent or revert morphometric alterations in proximal nerve portions in non-diabetic and diabetic injured rats.
PMCID: PMC3020345  PMID: 21340223
Diabetes; Sciatic nerve crush; Motor function; Nerve morphometry; Treadmill training
2.  The Use of Fiber-Reinforced Scaffolds Cocultured with Schwann Cells and Vascular Endothelial Cells to Repair Rabbit Sciatic Nerve Defect with Vascularization 
BioMed Research International  2013;2013:362918.
To explore the feasibility of biodegradable fiber-reinforced 3D scaffolds with satisfactory mechanical properties for the repair of long-distance sciatic nerve defect in rabbits and effects of vascularized graft in early stage on the recovery of neurological function, Schwann cells and vascular endothelial cells were cocultured in the fiber-reinforced 3D scaffolds. Experiment group which used prevascularized nerve complex for the repair of sciatic nerve defect and control group which only cultured with Schwann cells were set. The animals in both groups underwent electromyography to show the status of the neurological function recovery at 4, 8, and 16 weeks after the surgery. Sciatic nerve regeneration and myelination were observed under the light microscope and electron microscope. Myelin sheath thickness, axonal diameter, and number of myelinated nerve fiber were quantitatively analyzed using image analysis system. The recovery of foot ulcer, the velocity of nerve conduction, the number of regenerating nerve fiber, and the recovery of ultrastructure were increased in the experimental group than those in the control group. Prevascularized tissue engineered fiber-reinforced 3D scaffolds for the repair of sciatic nerve defects in rabbits can effectively promote the recovery of neurological function.
PMCID: PMC3893804  PMID: 24490158
3.  Conserved Dopamine Neurotrophic Factor-Transduced Mesenchymal Stem Cells Promote Axon Regeneration and Functional Recovery of Injured Sciatic Nerve 
PLoS ONE  2014;9(10):e110993.
Peripheral nerve injury (PNI) is a common disease that often results in axonal degeneration and the loss of neurons, ultimately leading to limited nerve regeneration and severe functional impairment. Currently, there are no effective treatments for PNI. In the present study, we transduced conserved dopamine neurotrophic factor (CDNF) into mesenchymal stem cells (MSCs) in collagen tubes to investigate their regenerative effects on rat peripheral nerves in an in vivo transection model. Scanning electron microscopy of the collagen tubes demonstrated their ability to be resorbed in vivo. We observed notable overexpression of the CDNF protein in the distal sciatic nerve after application of CDNF-MSCs. Quantitative analysis of neurofilament 200 (NF200) and S100 immunohistochemistry showed significant enhancement of axonal and Schwann cell regeneration in the group receiving CDNF-MSCs (CDNF-MSCs group) compared with the control groups. Myelination thickness, axon diameter and the axon-to fiber diameter ratio (G-ratio) were significantly higher in the CDNF-MSCs group at 8 and 12 weeks after nerve transection surgery. After surgery, the sciatic functional index, target muscle weight, wet weight ratio of gastrocnemius muscle and horseradish peroxidase (HRP) tracing demonstrated functional recovery. Light and electron microscopy confirmed successful regeneration of the sciatic nerve. The greater numbers of HRP-labeled neuron cell bodies and increased sciatic nerve index values (SFI) in the CDNF-MSCs group suggest that CDNF exerts neuroprotective effects in vivo. We also observed higher target muscle weights and a significant improvement in muscle atrophism in the CDNF-MSCs group. Collectively, these findings indicate that CDNF gene therapy delivered by MSCs is capable of promoting nerve regeneration and functional recovery, likely because of the significant neuroprotective and neurotrophic effects of CDNF and the superior environment offered by MSCs and collagen tubes.
PMCID: PMC4208796  PMID: 25343619
4.  Comparison of Morphometric Aspects of Light and Electron Microscopy of the Hypoglossal Nerve between Young and Aged Male Wistar Rats 
Cell Journal (Yakhteh)  2011;13(4):229-236.
Age-related changes occur in many different systems of the body. Many elderly people show dysphagia and dysphonia. This research was conducted to evaluate quantitatively the morphometrical changes of the hypoglossal nerve resulting from the aging process in young and aged rats.
Materials and Methods:
Through an experimental study ten male wistar rats (4 months: 5 rats, 24 months: 5 rats) were selected randomly from a colony of wistars in the UWC. After a fixation process and preparation of samples of the cervical portion of the hypoglossal nerve of these rats, light and electron microscopic imaging were performed. These images were evaluated according to the numbers and size of myelinated nerve fibers, nucleoli of Schwann cells, myelin sheath thickness, axon diameter, and g ratio. All data were analyzed by Mann-Whitney, a non-parametric statistical test.
In light microscope, numbers of myelinated nerve fibers, the mean entire nerve perimeters, the mean entire nerve areas and the mean entire nerve diameters in young and aged rats' were not significantly different between the two groups.
In electron microscope, numbers of myelinated axons, numbers of Schwann cell nucleoli and the mean g ratios of myelinated axon to Schwann cell in young and aged rats were not significantly different. The myelinated fiber diameters, the myelin sheath thicknesses, myelinated axon diameters and the mean g ratio of axon diameter to myelinated fiber diameter in young and aged fibers were significantly different
The mean g ratio of myelinated nerve fibers of peripheral nerves stabilizes at the level of 0.6 after maturation and persists without major change during adulthood. This ratio of axon diameter to fiber diameter (0.6) is optimum for normal conduction velocity of neural impulses. Our study indicated that the g ratio of myelinated nerve fiber of the hypoglossal nerve decreased prominently in aged rats and can be a cause of impairment in nerve function in old age. Thus, prospective studies concerning electrophysiological and conductive properties of the peripheral nerve could be useful to clarify further the effects of aging on peripheral nerves.
PMCID: PMC3584479  PMID: 23508137
Hypoglossal Nerve; Myelinated Nerve Fiber; Aging; Rat
5.  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.
PMCID: PMC3567071  PMID: 23409189
6.  Tacrolimus reduces scar formation and promotes sciatic nerve regeneration☆ 
Neural Regeneration Research  2012;7(32):2500-2506.
A sciatic nerve transection and repair model was established in Sprague-Dawley rats by transecting the tendon of obturator internus muscle in the greater sciatic foramen and suturing with nylon sutures. The models were treated with tacrolimus gavage (4 mg/kg per day) for 0, 2, 4 and 6 weeks. Specimens were harvested at 6 weeks of intragastric administration. Masson staining revealed that the collagen fiber content and scar area in the nerve anastomosis of the sciatic nerve injury rats were significantly reduced after tacrolimus administration. Hematoxylin-eosin staining showed that tacrolimus significantly increased myelinated nerve fiber density, average axon diameter and myelin sheath thickness. Intragastric administration of tacrolimus also led to a significant increase in the recovery rate of gastrocnemius muscle wet weight and the sciatic functional index after sciatic nerve injury. The above indices were most significantly improved at 6 weeks after of tacrolimus gavage. The myelinated nerve fiber density in the nerve anastomosis and the sciatic nerve functions had a significant negative correlation with the scar area, as detected by Spearman’s rank correlation analysis. These findings indicate that tacrolimus can promote peripheral nerve regeneration and accelerate the recovery of neurological function through the reduction of scar formation.
PMCID: PMC4200705  PMID: 25337101
tacrolimus; scar; myelinated nerve fiber; sciatic nerve; peripheral nerve injury; neural regeneration; neurological function
7.  In Vivo Serial Imaging of Regenerating Corneal Nerves after Surgical Transection in Transgenic Thy1-YFP mice 
After surgical transection, corneal nerves regenerate to achieve normal density but do not readopt the normal arrangement. Myelinated nerve fibers also regenerate along with nociceptive nerve fibers in the central corneal stroma.
To determine the effect of lamellar transection surgery on the nerve fiber density (NFD) and pattern of nerve regeneration in the cornea of thy1-YFP transgenic mice.
Wide-field stereo fluorescence microscopy was used to obtain serial images of nerves in live thy1-YFP mice, which express a fluorescent protein in their axons. NFD (mm/mm2) was calculated from maximum intensity projection images as the total length of fibers within the area of the contour in which nerves were traced. Whole-mount confocal microscopy was performed to analyze the arrangement of nerves and the types of regenerating fibers.
NFD in normal corneas was 35.3 ± 1.8 mm/mm2. Stereo fluorescence microscopy revealed the presence of a subbasal hairpin nerve layer and an intrastromal nerve trunk layer. After surgery, regenerative sprouting was observed from transected distal ends of intrastromal nerve trunks. NFD also increased, with this increase being maximal between 4 and 6 weeks after surgery. NFD approximated baseline values at 6 weeks and did not change any further at 8 weeks. Regenerated nerves did not readopt the normal corneal nerve arrangement. A dense interlacing network of regenerated nerves was present in the corneal bed. Branches from this network traversed the flap to innervate the epithelium. Immunofluorescence staining revealed that regenerating fronds contained peptidergic nociceptive fibers (positive for calcitonin gene-related peptide and substance P) and myelinated non-nociceptive fibers (positive for neurofilament 200).
Although corneal NFD recovers to normal levels by 8 weeks after nerve transection, the arrangement of regenerated nerves is abnormal.
PMCID: PMC3207999  PMID: 21896845
8.  Impaired Prosaposin Secretion During Nerve Regeneration in Diabetic Rats and Protection of Nerve Regeneration by a Prosaposin-Derived Peptide 
Prosaposin is both a precursor of sphingolipid activator proteins and a secreted neurotrophic and myelinotrophic factor. Because peripheral nerve regeneration is impaired in diabetes mellitus, we measured prosaposin protein levels from control and streptozotocin-diabetic rats by collecting endoneurial fluid secreted into a bridging tube connecting the ends of transected sciatic nerve. Prosaposin protein levels were significantly reduced in endoneurial fluid from diabetic rats and increased in the proximal nerve stump compared to controls. To investigate whether a prosaposin-derived peptide could improve nerve regeneration, rats were treated with prosaptide TX14(A) following sciatic nerve crush. In control rats, TX14(A) was without effect in the uninjured nerve but shortened toe spread recovery time after nerve crush. In diabetic rats, efficacy of prosaptide TX14(A) was confirmed by correction of thermal hypoalgesia, formalin-evoked hyperalgesia and conduction slowing in the uninjured nerve. The peptide also prevented diabetes-induced abnormalities in nerve regeneration distance and mean axonal diameter of regenerated axons, whereas delayed recovery of toe spread was not improved. Muscle denervation atrophy was attenuated by TX14(A) in both control and diabetic rats. These results suggest that reduced prosaposin secretion after nerve injury may contribute to impaired regeneration rates in diabetic rats and that prosaptide TX14(A) can improve aspects of nerve regeneration.
PMCID: PMC2748883  PMID: 18596543
Diabetes; Nerve regeneration; Prosaposin; Prosaptide TX14(A)
A correlation of the histopathology and clinical behavior of thiamin deficient pigeons has been undertaken. Opisthotonus in acutely deficient pigeons was frequently attended by no degenerating nerve fibers or neurons in either the central or peripheral nervous systems. When the deficiency was complicated by starvation, it developed more slowly, opisthotonus appeared later, and many degenerating nerve fibers were usually present. In both instances the opisthotonus disappeared in a very short time after thiamin was injected intramuscularly. A more chronic deficiency, characterized by leg weakness (opisthotonus being absent) appeared when the ration was partially deficient in thiamin, or occasionally when the caloric intake was grossly inadequate. In birds of this type degenerating nerve fibers were always found in the peripheral nerves. The number of such fibers in the sciatic nerves corresponded closely with the degree of paralysis, and during repair which occurred when thiamin (irrespective of other factors) was added to the ration, nerve fibers regenerated (increased in number) at a rate which paralleled the clinical improvement closely. The large and long nerve fibers, many of which could be traced directly into the dorsal ganglia, degenerated first, and if the deficiency were prolonged, smaller nerve fibers became affected as well. In many of these pigeons with marked leg weakness, cell bodies in the dorsal ganglia exhibited lysis of chromatin and eccentricity of their nuclei. This was observed nowhere else in the nervous system. During repair and until after the paralysis had been recovered from completely, these phenomena (chromatolysis) persisted. In chronically thiamin deficient pigeons large and long degenerating nerve fibers were found in two regions of the spinal cord at all levels. One group of these in the ventral funiculus was thought to arise in the reticular region of the medulla oblongata, and the other which was situated in the posterior part of the lateral funiculus could be followed to the lateral surface of the medulla oblongata, and from there by way of the inferior cerebellar peduncles into the medullary portion of the cerebellum. In the central as well as the peripheral nervous system the long and large nerve fibers degenerated first. Medium and small sized fibers were affected later and the degeneration became quite generalized. In many of the chronically deficient pigeons with leg weakness, incidental postmortem findings compatible with cardiac failure were encountered. In the hearts from many of these pigeons, microscopic examination revealed many areas of focal necrosis, some of which had become infiltrated with polymorphonuclear leucocytes. In a peripheral neuron of a thiamin deficient pigeon the first consistent morphological alteration appeared in the axis cylinder. No doubt a period of functional impairment of the neuron (such as produced opisthotonus) preceded this. The axis cylinder followed by the myelin sheath degenerated at a point most distal to its trophic cell body. This process of disintegration proceeded toward the trophic cell body for a variable distance, depending upon the severity and duration of the deficiency. The cell body (in a dorsal ganglion) appeared to shrink first and later exhibited chromatolysis. When thiamin was administered the axis cylinder (and myelin sheath) regenerated, and when this was complete, the cell body returned to normal. It has been concluded that the opisthotonus of thiamin deficiency is a manifestation of decerebration due to a functional impairment of the neurons which have an inhibitory influence upon the lower brain stem centers. Leg weakness (when produced by the same deficiency) is due to degeneration of peripheral nerve fibers within the sciatic nerve. Heart failure may be attended by no visible histological changes, but in many instances necrosis of myocardial fibers occurs.
PMCID: PMC2135092  PMID: 19870991
10.  Electrical Stimulation Promotes Regeneration of Defective Peripheral Nerves after Delayed Repair Intervals Lasting under One Month 
PLoS ONE  2014;9(9):e105045.
Electrical stimulation (ES) has been proven to be an effective means of enhancing the speed and accuracy of nerve regeneration. However, these results were recorded when the procedure was performed almost immediately after nerve injury. In clinical settings, most patients cannot be treated immediately. Some patients with serious trauma or contaminated wounds need to wait for nerve repair surgery. Delays in nerve repair have been shown to be associated with poorer results than immediate surgery. It is not clear whether electrical stimulation still has any effect on nerve regeneration after enough time has elapsed.
A delayed nerve repair model in which the rats received delayed nerve repair after 1 day, 1 week, 1 month, and 2 months was designed. At each point in time, the nerve stumps of half the rats were bridged with an absorbable conduit and the rats were given 1 h of weak electrical stimulation. The other half was not treated. In order to analyze the morphological and molecular differences among these groups, 6 ES rats and 6 sham ES rats per point in time were killed 5 days after surgery. The other rats in each group were allowed to recover for 6 weeks before the final functional test and tissue observation.
The amounts of myelinated fibers in the distal nerve stumps decreased as the delay in repair increased for both ES rats and sham ES rats. In the 1-day-delay and 1-week-delay groups, there were more fibers in ES rats than in sham ES rats. And the compound muscle action potential (CMAP) and motor nerve conduction velocity (MNCV) results were better for ES rats in these two groups. In order to analyze the mechanisms underlying these differences, Masson staining was performed on the distal nerves and quantitative PCR on the spinal cords. Results showed that, after delays in repair of 1 month and 2 months, there was more collagen tissue hyperplasia in the distal nerve in all rats. The brain-derived neurotrophic factor (BDNF) and trkB expression levels in the spinal cords of ES rats were higher than in sham ES rats. However, these differences decreased as the delay in repair increased.
Electrical stimulation does not continue to promote nerve regeneration after long delays in nerve repair. The effective interval for nerve regeneration after delayed repair was found to be less than 1 month. The mechanism seemed to be related to the expression of nerve growth factors and regeneration environment in the distal nerves.
PMCID: PMC4152131  PMID: 25181499
11.  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.
PMCID: PMC3595679  PMID: 23533510
12.  Use of Natural Neural Scaffolds Consisting of Engineered Vascular Endothelial Growth Factor Immobilized on Ordered Collagen Fibers Filled in a Collagen Tube for Peripheral Nerve Regeneration in Rats 
The search for effective strategies for peripheral nerve regeneration has attracted much attention in recent years. In this study, ordered collagen fibers were used as intraluminal fibers after nerve injury in rats. Vascular endothelial growth factor (VEGF) plays an important role in nerve regeneration, but its very fast initial burst of activity within a short time has largely limited its clinical use. For the stable binding of VEGF to ordered collagen fibers, we fused a collagen-binding domain (CBD) to VEGF through recombinant DNA technology. Then, we filled the ordered collagen fibers-CBD-VEGF targeting delivery system in a collagen tube to construct natural neural scaffolds, which were then used to bridge transected nerve stumps in a rat sciatic nerve transection model. After transplantation, the natural neural scaffolds showed minimal foreign body reactions and good integration into the host tissue. Oriented collagen fibers in the collagen tube could guide regenerating axons in an oriented manner to the distal, degenerating nerve segment, maximizing the chance of target reinnervation. Functional and histological analyses indicated that the recovery of nerve function in the natural neural scaffolds-treated group was superior to the other grafted groups. The guiding of oriented axonal regeneration and effective delivery systems surmounting the otherwise rapid and short-lived diffusion of growth factors in body fluids are two important strategies in promoting peripheral nerve regeneration. The natural neural scaffolds described take advantage of these two aspects and may produce synergistic effects. These properties qualified the artificial nerve conduits as a putative candidate system for the fabrication of peripheral nerve reconstruction devices.
PMCID: PMC4227234  PMID: 25322152
engineered vascular endothelial growth factor (VEGF); natural neural scaffolds; biodebradable materials; peripheral nerve regeneration
13.  Impaired nerve fiber regeneration in axotomized peripheral nerves in streptozotocin‐diabetic rats 
Impaired nerve fiber regeneration is a salient feature of diabetic neuropathy. Its pathogenesis is still unclear. We attempted to characterize the structure of regenerated myelinated fibers after transection in streptozotocin‐diabetic rats.
Materials and Methods
Streptozotocin‐diabetic rats underwent transection of the sciatic nerve. Two and 4 weeks post‐axotomy, regenerated myelinated fibers of the cut end and fibers at its proximal site were morphometrically examined. Non‐diabetic control rats with axotomy were also examined for comparison.
At 4 weeks post‐axotomy, diabetic rats showed an increased myelinated fiber density and total fiber number with a trend toward reduced fiber size at the cut end compared with those in control rats. The average number of myelin lamellae relative to axonal size in regenerated fibers at the cut end was significantly reduced in diabetic rats compared with that in control rats. The proximal site showed a reduced size of fibers and axons in both diabetic and control rats to a similar extent compared with those in a non‐axotomized state. At 2 weeks post‐axotomy, these findings were less apparent.
The nerves of diabetic rats when axotomized undergo impaired regeneration characterized by increased fiber density with hypomyelination.
PMCID: PMC4020247  PMID: 24843706
Fiber atrophy; Myelination; Nerve regeneration
14.  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.
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
15.  Effect of limb lengthening on internodal length and conduction velocity of peripheral nerve 
The influences of axon diameter, myelin thickness and internodal length on the velocity of conduction of peripheral nerve action potentials are unclear. Previous studies have demonstrated a strong dependence of conduction velocity on internodal length. However, a theoretical analysis has suggested that this relationship may be lost above a nodal separation of about 0.6 mm. Here we measured nerve conduction velocities in a rabbit model of limb lengthening that produced compensatory increases in peripheral nerve growth. Divided tibial bones in one hind limb were gradually lengthened at 0.7 mm per day using an external frame attached to the bone. This was associated with a significant increase (33%) of internodal length (0.95 to 1.3 mm) in axons of the tibial nerve that varied in proportion to the mechanical strain in the nerve of the lengthened limb. Axonal diameter, myelin thickness and g-ratios were not significantly altered by limb-lengthening. Despite the substantial increase in internodal length, no significant change was detected in conduction velocity (about 43 ms-1) measured either in vivo or in isolated tibial nerves. The results demonstrate that the internode remains plastic in the adult but that increases in internodal length of myelinated adult nerve axons do not result in either deficiency or proportionate increases in their conduction velocity and supports the view that the internodal lengths of nerves reach a plateau beyond which their conduction velocities are no longer sensitive to increases in internodal length.
PMCID: PMC4335134  PMID: 23467369
16.  Effects of Valproic Acid on Axonal Regeneration and Recovery of Motor Function after Peripheral Nerve Injury in the Rat 
Valproic acid (VPA) is used to be an effective anti-epileptic drug and mood stabilizer. It has recently been demonstrated that VPA could promote neurite outgrowth, activate the extracellular signal regulated kinase pathway, and increases bcl-2 and growth cone-associated protein 43 levels in spinal cord. In the present research we demonstrate the effect of VPA on peripheral nerve regeneration and recovery of motor function following sciatic nerve transaction in rats.
The rats in VPA group and control group were administered with valproic acid (300mg/kg) and sodium chloride respectively after operation. Each animal was observed sciatic nerve index (SFI) at 2-week intervals and studied electrophysiology at 4-week intervals for 12 weeks. Histological and morphometrical analyses were performed 12 weeks after operation. Using the digital image-analysis system, thickness of the myelin sheath was measured, and total numbers of regenerated axons were counted.
There was a significant difference in SFI, electrophysiological index (motor-nerve conduct velocity), and morphometrical results (regenerated axon number and thickness of myelin sheath) in nerve regeneration between the VPA group and controls (P<0.05).
The results demonstrated that VPA is able to enhance sciatic nerve regeneration in rats, suggesting the potential clinical application of VPA for the treatment of peripheral nerve injury in humans.
PMCID: PMC4151439  PMID: 25207308
Bcl-2; Growth cone-associated protein 43; Myelin; Rat; Sciatic nerve index; Valproic acid
17.  “Supercharge Nerve Transfer to Enhance Motor Recovery, a Laboratory Study” 
The Journal of hand surgery  2013;38(3):466-477.
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.
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.
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.
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.
PMCID: PMC3583195  PMID: 23391355
Nerve regeneration; nerve transfer; neurorrhaphy; peripheral nerve; supercharge end-to-side
The Journal of General Physiology  1951;35(1):145-182.
Using the ability of the nerve fibers to conduct impulses as indicator of changes in the concentration of sodium ions in the interstitial spaces of nerve an evaluation has been made of the diffusion constant of sodium ions. The calculated minimal value (0.62 x 10–4 cm.2/min.) undoubtedly is much too low; nevertheless, it is still so high that as a rule the diffusion of sodium ions is far more rapid than the establishment of excitability changes; therefore, diffusion times need not be taken into account in the interpretation of ordinary experiments. By measurements of the changes in the longitudinal conductivity of nerve which result from changes in the external concentration of sodium chloride an evaluation has been made of the diffusion constant of sodium chloride in the interstitial spaces of nerve. A minimal value for this constant is 1.4 x 10–4 cm.2/min. The evidence presented would be compatible with the assumption that the permeability of the connective tissue sheath for sodium ions decreases slightly after the concentration of sodium ions in the interstitial spaces of the nerve has become negligible; the evidence, however, shows that changes in the permeability of the sheath cannot play a significant role in determining the temporal courses of the development of inexcitability in a sodium-free medium and of the restoration of excitability by added sodium ions. If a decrease in the permeability of the sheath should take place in a sodium-free medium, the change would be small and would occur after the nerve fibers have become inexcitable; on the other hand the action of a moderate concentration of sodium ions would be sufficient to restore the permeability of the sheath. As measured by the recovery by A fibers of the ability to conduct impulses the restoration by 0.1 N sodium ions of nerve that has been deprived of sodium for 15 to 20 hours, i.e. for several hours after the nerve fibers have become inexcitable, begins after a significant delay, since no A fiber begins to conduct impulses in less than 8 or 10 minutes. The delay is referable to the fact that, before the A fibers can regain the ability to conduct impulses, those changes in their properties have to be reversed, which have taken place in the absence of sodium ions. Usually within 1 minute after sodium ions are made available to the nerve the polarizability of the membrane by the anodal current begins to increase; the A fibers soon begin to produce unconducted impulses in response to the break of the anodal current; then, they produce unconducted impulses in response to the closure of the cathodal current, and finally they become able to conduct impulses, although at a markedly reduced speed. The C fibers, that become inexcitable in a sodium-free medium later than the A fibers, begin to conduct impulses within 1 minute or 2 after 0.1 N sodium ions are made available to the nerve. Treatment of a nerve, that has been kept in a sodium-free medium, for 15 to 20 hours, with a moderate concentration of sodium ions (0.015, 0.02 N), acting for 1 hour or 2, is not sufficient to restore the ability to conduct impulses to more than a few A fibers, but it produces in a relatively large number of fibers a partial restoration, so that when the concentration of sodium ions outside the epineurium is increased by 0.005 or 0.01 N a significant number of A fibers begin to conduct impulses within less than 5 seconds. Initially the recovery progresses with great rapidity, but after a small number of minutes the height of the conducted spike remains practically stationary. Increase of the external concentration of sodium ions by a small amount again causes a rapid enhancement of the recovery, but once more, after a few minutes the height of the spike remains practically stationary, etc. A subnormal concentration of sodium ions may restore to all the A fibers the ability to conduct impulses, but only 0.1 N sodium ions are able to produce a complete restoration of the speed of conduction, and only after they have been allowed to act for a considerable period of time. The ability of all the C fibers to conduct impulses may be restored by relatively small concentrations of sodium ions, 0.02 to 0.025 N. Nerve fibers that have become inexcitable in a sodium-free medium and have been restored by sodium ions are far more sensitive to the effect of the lack of sodium than the fibers of untreated nerve. Repeated removal and addition of sodium ions may bring the nerve fibers, especially those of spinal roots, to a state in which the sensitivity to the lack of sodium is exceedingly great; spinal root fibers may then begin to become inexcitable in a sodium-free medium within a few seconds. Treatment of the nerve with 0.1 N sodium ions for 1 hour or 2 is sufficient to bring about a marked increase in the resistance to the lack of sodium. On the other hand keeping a nerve in Ringer's solution or in the presence of 0.04 N sodium ions does not produce a readily detectable increase in the sensitivity to the lack of sodium. Even the resistance of nerve kept in the presence of 0.025 N sodium ions for 23 hours is very high, since after 2 hours in a sodium-free medium more than two-thirds of the initially conducting fibers will be able to conduct impulses. Frog nerve reaches different states of equilibrium with different external concentrations of sodium ions. The states are characterized by the degree of effectiveness of the nerve reaction, the speed of conduction of impulses, and the number of conducting fibers. Approximately the same equilibrium state may be reached by (a) leaving the nerve for 20 to 24 hours in the presence of a subnormal concentration of sodium ions and (b) by leaving the nerve in a sodium-free medium for 15 to 20 hours, restoring it with 0.1 N sodium ions acting for a short period of time, rendering it inexcitable again in a sodium-free medium, and finally restoring it with a moderate concentration of sodium ions. If, however, the nerve that has been kept in a sodium-free medium for 15 to 20 hours is restored directly by a moderate concentration of sodium ions the state will not be reached, at least not for several hours, which corresponds to equilibrium with that concentration. The role of sodium in nerve physiology is discussed. Sodium participates in at least four processes, (a) The regulation of the concentration of water outside the nerve fibers; (b) the regulation of the total value of the membrane potential; (c) the production of the nerve impulse, and (d) the establishment of the nerve reaction. In so far as processes (c) and (d) are concerned only the sodium present inside the nerve fibers plays a role; the presence of sodium ions outside the nerve fibers is important only because in the absence of interstitial sodium ions the nerve fibers lose a part of their internal sodium content. The nerve impulse and the nerve reaction may be produced for long periods of time after the concentration of sodium ions outside the nerve fibers has become negligible. A working hypothesis is put forward according to which the internal sodium content and the interstitial concentration of sodium ions are in equilibrium in so far as a different internal sodium content corresponds to each interstitial concentration. The properties of the nerve fibers are determined by the internal sodium content. The change in properties, i.e. in the state of the nerve fibers, results from processes that take place inside the nerve fibers after the interstitial concentration of sodium ions and consequently also the internal sodium content have been changed.
PMCID: PMC2147304  PMID: 14873926
The Journal of General Physiology  1955;38(5):709-728.
As an aid in the interpretation of the physiological properties of unmedullated nerve fibers, particularly those having their cells of origin in the dorsal root ganglia, more precise information about their morphology has been acquired through employment of the electron microscope. The appearance of the fibers in the skin nerves is described, with special reference to the structure of their sheaths; and a notation is made about the bearing of the axon-sheath relationship on the biophysical mechanism of conduction (p. 714). There is no basic difference between the sheath systems of the d.r.C and the s.C fibers. Attention is called to a point of similarity between the sheaths of unmyelinated and myelinated axons (p. 715). An assessment was made of the likelihood of interaction between the fibers. In action potentials showing temporal dispersion at several distances, the elevations appeared in their calculated positions. A model of a group of Schwann sheaths was constructed from successive electron microscope sections, showing that the lengths of the sheath branches are short in comparison with the wave lengths of the action potentials. Supported by these and other considerations, the argument is strongly in favor of the conclusion that among d.r.C fibers, as in other fibers, there is no cross-excitation between the axons. A new analysis of the size distribution of the fibers in a sural nerve was made from electron microscope pictures; and from the measurements the action potential was constructed. The result confirmed the view, previously expressed, that the velocities of conduction in the fibers can be precisely accounted for by multiplying the diameters by a constant. In the dorsal roots, the striking change that takes place in the appearance of the fibers and their disposition in the Schwann sheaths can be seen in Fig. 11. The axons partake of the special properties of the peripheral branches, which necessitated the creation of the subdivision of d.r.C fibers. But, their diameters are much smaller. At a set of reduced conduction velocities the configuration of the compound action potential in the nerves is repeated in the roots, with the root velocities still conforming to the size-velocity rule derived from nerve axons.
PMCID: PMC2147503  PMID: 14367780
20.  Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration 
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.
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.
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.
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.
PMCID: PMC3465232  PMID: 22776032
RGD peptide; Nerve growth factor; Peripheral nerve; Nerve conduits; Nerve regeneration
21.  Chemoattractive capacity of different lengths of nerve fragments bridging regeneration chambers for the repair of sciatic nerve defects 
Neural Regeneration Research  2012;7(29):2293-2298.
A preliminary study by our research group showed that 6-mm-long regeneration chamber bridging is equivalent to autologous nerve transplantation for the repair of 12-mm nerve defects. In this study, we compared the efficacy of different lengths (6, 8, 10 mm) of nerve fragments bridging 6-mm regeneration chambers for the repair of 12-mm-long nerve defects. At 16 weeks after the regeneration chamber was implanted, the number, diameter and myelin sheath thickness of the regenerated nerve fibers, as well as the conduction velocity of the sciatic nerve and gastrocnemius muscle wet weight ratio, were similar to that observed with autologous nerve transplantation. Our results demonstrate that 6-, 8- and 10-mm-long nerve fragments bridging 6-mm regeneration chambers effectively repair 12-mm-long nerve defects. Because the chemoattractive capacity is not affected by the length of the nerve fragment, we suggest adopting 6-mm-long nerve fragments for the repair of peripheral nerve defects.
PMCID: PMC4268731  PMID: 25538752
nerve fragment length; nerve regeneration chamber; bridging; long nerve defect; chemotactic ability; neural regeneration
22.  Peptide Mimetic of the S100A4 Protein Modulates Peripheral Nerve Regeneration and Attenuates the Progression of Neuropathy in Myelin Protein P0 Null Mice 
Molecular Medicine  2013;19(1):43-53.
We recently found that S100A4, a member of the multifunctional S100 protein family, protects neurons in the injured brain and identified two sequence motifs in S100A4 mediating its neurotrophic effect. Synthetic peptides encompassing these motifs stimulated neuritogenesis and survival in vitro and mimicked the S100A4-induced neuroprotection in brain trauma. Here, we investigated a possible function of S100A4 and its mimetics in the pathologies of the peripheral nervous system (PNS). We found that S100A4 was expressed in the injured PNS and that its peptide mimetic (H3) affected the regeneration and survival of myelinated axons. H3 accelerated electrophysiological, behavioral and morphological recovery after sciatic nerve crush while transiently delaying regeneration after sciatic nerve transection and repair. On the basis of the finding that both S100A4 and H3 increased neurite branching in vitro, these effects were attributed to the modulatory effect of H3 on initial axonal sprouting. In contrast to the modest effect of H3 on the time course of regeneration, H3 had a long-term neuroprotective effect in the myelin protein P0 null mice, a model of dysmyelinating neuropathy (Charcot-Marie-Tooth type 1 disease), where the peptide attenuated the deterioration of nerve conduction, demyelination and axonal loss. From these results, S100A4 mimetics emerge as a possible means to enhance axonal sprouting and survival, especially in the context of demyelinating neuropathies with secondary axonal loss, such as Charcot-Marie-Tooth type 1 disease. Moreover, our data suggest that S100A4 is a neuroprotectant in PNS and that other S100 proteins, sharing high homology in the H3 motif, may have important functions in PNS pathologies.
PMCID: PMC3646097  PMID: 23508572
23.  Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and myelin-associated glycoprotein) in regenerating adult mouse sciatic nerve 
The Journal of Cell Biology  1988;106(5):1735-1746.
The localization of the neural cell adhesion molecules L1, N-CAM, and the myelin-associated glycoprotein was studied by pre- and postembedding staining procedures at the light and electron microscopic levels in transected and crushed adult mouse sciatic nerve. During the first 2-6 d after transection, myelinated and nonmyelinated axons degenerated in the distal part of the proximal stump close to the transection site and over the entire length of the distal part of the transected nerve. During this time, regrowing axons were seen only in the proximal, but not in the distal nerve stump. In most cases L1 and N- CAM remained detectable at cell contacts between nonmyelinating Schwann cells and degenerating axons as long as these were still morphologically intact. Similarly, myelin-associated glycoprotein remained detectable in the periaxonal area of the degenerating myelinated axons. During and after degeneration of axons, nonmyelinating Schwann cells formed slender processes which were L1 and N-CAM positive. They resembled small-diameter axons but could be unequivocally identified as Schwann cells by chronical denervation. Unlike the nonmyelinating Schwann cells, only few myelinating ones expressed L1 and N-CAM. At the cut ends of the nerve stumps a cap developed (more at the proximal than at the distal stump) that contained S-100-negative and fibronectin-positive fibroblast-like cells. Most of these cells were N-CAM positive but always L1 negative. Growth cones and regrowing axons expressed N-CAM and L1 at contact sites with these cells. Regrowing axons of small diameter were L1 and N- CAM positive where they made contact with each other or with Schwann cells, while large-diameter axons were only poorly antigen positive or completely negative. 14 d after transection, when regrowing axons were seen in the distal part of the transected nerve, regrowing axons made L1- and N-CAM-positive contacts with Schwann cells. When contacting basement membrane, axons were rarely found to express L1 and N-CAM. Most, if not all, Schwann cells associated with degenerating myelin expressed L1 and N-CAM. In crushed nerves, the immunostaining pattern was essentially the same as in the cut nerve. During formation of myelin, the sequence of adhesion molecule expression was the same as during development: L1 disappeared and N-CAM was reduced on myelinating Schwann cells and axons after the Schwann cell process had turned approximately 1.5 loops around the axon. Myelin-associated glycoprotein then appeared both periaxonally and on the turning loops of Schwann cells in the uncompacted myelin.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC2115039  PMID: 2453520
24.  Expression of ATF3 and axonal outgrowth are impaired after delayed nerve repair 
BMC Neuroscience  2008;9:88.
A delay in surgical nerve repair results in impaired nerve function in humans, but mechanisms behind the weakened nerve regeneration are not known. Activating transcription factor 3 (ATF3) increases the intrinsic growth state of injured neurons early after injury, but the role of long-term changes and their relation to axonal outgrowth after a delayed nerve repair are not well understood. ATF3 expression was examined by immunohistochemistry in motor and sensory neurons and in Schwann cells in rat sciatic nerve and related to axonal outgrowth after transection and delayed nerve repair (repair 0, 30, 90 or 180 days post-injury). Expression of the neuronal cell adhesion molecule (NCAM), which is expressed in non-myelinating Schwann cells, was also examined.
The number of neurons and Schwann cells expressing ATF3 declined and the length of axonal outgrowth was impaired if the repair was delayed. The decline was more rapid in motor neurons than in sensory neurons and Schwann cells. Regeneration distances over time correlated to number of ATF3 stained neurons and Schwann cells. Many neurofilament stained axons grew along ATF3 stained Schwann cells. If nerve repair was delayed the majority of Schwann cells in the distal nerve segment stained for NCAM.
Delayed nerve repair impairs nerve regeneration and length of axonal outgrowth correlates to ATF3 expression in both neurons and Schwann cells. Mainly non-myelinating Schwann cells (NCAM stained) are present in distal nerve segments after delayed nerve repair. These data provide a neurobiological basis for the poor outcomes associated with delayed nerve repair. Nerve trunks should, if possible, be promptly repaired.
PMCID: PMC2556676  PMID: 18801180
25.  Early changes in muscle atrophy and muscle fiber type conversion after spinal cord transection and peripheral nerve transection in rats 
Spinal cord transection and peripheral nerve transection cause muscle atrophy and muscle fiber type conversion. It is still unknown how spinal cord transection and peripheral nerve transection each affect the differentiation of muscle fiber type conversion mechanism and muscle atrophy. The aim of our study was to evaluate the difference of muscle weight change, muscle fiber type conversion, and Peroxisome proliferator-activated receptor-γ coactivatior-1α (PGC-1α) expression brought about by spinal cord transection and by peripheral nerve transection.
Twenty-four Wistar rats underwent surgery, the control rats underwent a laminectomy; the spinal cord injury group underwent a spinal cord transection; the denervation group underwent a sciatic nerve transection. The rats were harvested of the soleus muscle and the TA muscle at 0 week, 1 week and 2 weeks after surgery. Histological examination was assessed using hematoxylin and eosin (H&E) staining and immunofluorescent staing. Western blot was performed with 3 groups.
Both sciatic nerve transection and spinal cord transection caused muscle atrophy with the effect being more severe after sciatic nerve transection. Spinal cord transection caused a reduction in the expression of both sMHC protein and PGC-1α protein in the soleus muscle. On the other hand, sciatic nerve transection produced an increase in expression of sMHC protein and PGC-1α protein in the soleus muscle. The results of the expression of PGC-1α were expected in other words muscle atrophy after sciatic nerve transection is less than after spinal cord transection, however muscle atrophy after sciatic nerve transection was more severe than after spinal cord transection.
In the conclusion, spinal cord transection diminished the expression of sMHC protein and PGC-1α protein in the soleus muscle. On the other hand, sciatic nerve transection enhanced the expression of sMHC protein and PGC-1α protein in the soleus muscle.
PMCID: PMC3668998  PMID: 23687941
Muscle atrophy; Muscle fiver type conversion; Spinal cord transection; Peripheral nerve transection; Rat

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