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1.  Impaired nerve fiber regeneration in axotomized peripheral nerves in streptozotocin‐diabetic rats 
Abstract
Aim/Introduction
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.
Results
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.
Conclusions
The nerves of diabetic rats when axotomized undergo impaired regeneration characterized by increased fiber density with hypomyelination.
doi:10.1111/jdi.12115
PMCID: PMC4020247  PMID: 24843706
Fiber atrophy; Myelination; Nerve regeneration
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.
doi:10.1155/2013/362918
PMCID: PMC3893804  PMID: 24490158
3.  “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
4.  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
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.
doi:10.1371/journal.pone.0056484
PMCID: PMC3567071  PMID: 23409189
6.  Diet Induced Obesity in Sprague Dawley Rats Causes Microvascular and Neural Dysfunction 
Background
The objective was to determine the effect of diet induced obesity (DIO) on microvascular and neural function.
Methods
Rats were fed a standard or high fat diet for up to 32 weeks. Measurements were performed of vasodilation in epineurial arterioles by videomicroscopy, endoneurial blood flow by hydrogen clearance, nerve conduction velocity by electrical stimulation, size-frequency distribution of myelinated fibers of the sciatic nerve, intraepidermal nerve fiber density using confocal microscopy and thermal nociception using the Hargreaves method.
Results
Rats fed a high fat diet for 32 weeks developed sensory neuropathy as indicated by slowing of sensory nerve conduction velocity and thermal hypoalgesia. Motor nerve conduction velocity and endoneurial blood flow were not impaired. Mean axonal diameter of myelinated fibers of the sciatic nerve was unchanged in high fat fed rats compared to control. Intraepidermal nerve fiber density was significantly reduced in high fat fed rats. Vascular relaxation to acetylcholine and calcitonin gene-related peptide was decreased and expression of neutral endopeptidase (NEP) increased in epineurial arterioles of rats fed a high fat diet. In contrast, insulin-mediated vascular relaxation was increased in epineurial arterioles. NEP activity was significantly increased in the skin of the hindpaw. Markers of oxidative stress were increased in the aorta and serum of high fat fed rats but not in epineurial arterioles.
Conclusion
Chronic obesity causes microvascular and neural dysfunction. This is associated with increased expression of NEP but not oxidative stress in epineurial arterioles. NEP degrades vasoactive peptides which may explain the decrease in microvascular function.
doi:10.1002/dmrr.1088
PMCID: PMC2878284  PMID: 20503263
Obesity; vascular function; neutral endopeptidase; neuropathy
7.  An implantable electrical stimulator used for peripheral nerve rehabilitation in rats 
This study evaluated an implantable electrical stimulator using a sciatic nerve injury animal model, and ethological, electrophysiological and histological assessments. Forty Sprague-Dawley rats were used in the study, and were subjected to crushing of the right sciatic nerve with a micro-vessel clamp. Electrical stimulators were implanted in twenty of the rats (the implantation group), while the remaining twenty rats were assigned to the control group. At three and six weeks following the surgery, the sciatic nerve function index (SFI) and the motor nerve conduction velocity (MCV) were demonstrated to be significantly higher in the implantation group compared with the control group (P<0.05). Histological analysis, using hematoxylin and eosin (H&E) staining, showed the typical pathological atrophy, and an assessment of the nerve that had been crushed revealed distal axonal breakdown in the control group. These results suggest that the implantable electrical stimulator was effective, and was suitable for implantation in a Sprague-Dawley rat model.
doi:10.3892/etm.2013.1110
PMCID: PMC3735806  PMID: 23935712
implantable stimulator; animal model; electrical stimulation; peripheral nerve injury
8.  The Multiple Silicone Tube Device, “Tubes within a Tube,” for Multiplication in Nerve Reconstruction 
BioMed Research International  2014;2014:689127.
Multiple nerve branches were created during the regeneration procedure after a nerve injury and such multiple branches are suggested to be used to control, for example, prosthesis with many degrees of freedom. Transected rat sciatic nerve stumps were inserted into a nine mm long silicone tube, which contained four, five mm long, smaller tubes, thus leaving a five mm gap for regenerating nerve fibers. Six weeks later, several new nerve structures were formed not only in the four smaller tubes, but also in the spaces in-between. The 7–9 new continuous nerve structures, which were isolated as individual free nerves after removal of the tubes, were delineated by a perineurium and contained both myelinated and unmyelinated nerve fibers as well as blood vessels. Stimulation of the proximal nerve elicited contractions in distal muscles. Thin metal electrodes, inserted initially into the smaller tubes in some experiments, became embedded in the new nerve structures and when stimulated contractions of the distal muscles were observed. The “tubes within a tube” technique, creating multiple new nerves from a single “mother” nerve, can be used to record multiple signals for prosthetic device control or as sources for supply of multiple denervated targets.
doi:10.1155/2014/689127
PMCID: PMC4016851  PMID: 24864255
9.  Selectivity in the Reinnervation of the Lateral Gastrocnemius Muscle after Nerve Repair with Ethyl Cyanoacrylate in the Rat 
There is a need for complementary surgical techniques that enable rapid and reliable primary repair of transected nerves. Previous studies after peripheral nerve transection and repair with synthetic adhesives have demonstrated regeneration to an extent comparable to that of conventional techniques. The aim of this study was to compare two different repair techniques on the selectivity of muscle reinnervation after repair and completed regeneration. We used the cholera toxin B technique of retrograde axonal tracing to evaluate the morphology, the number, and the three-dimensional location of α-motoneurons innervating the lateral gastrocnemius muscle and compared the results after repair with either ethyl cyanoacrylate (ECA) or epineural sutures of the transected parent sciatic nerve. In addition, we recorded the wet weight of the muscle. Six months after transection and repair of the sciatic nerve, the redistribution of the motoneuron pool was markedly disorganized, the motoneurons had apparently increased in number, and they were scattered throughout a larger volume of the spinal cord gray matter with a decrease in the synaptic coverage compared to controls. A reduction in muscle weight was observed as well. No difference in morphometric variables or muscle weight between the two repair methods could be detected. We conclude that the selectivity of motor reinnervation following sciatic nerve transection and subsequent repair with ECA is comparable to that following conventional micro suturing.
doi:10.3389/fneur.2011.00025
PMCID: PMC3087165  PMID: 21577248
nerve repair; cyanoacrylate; synthetic adhesive; retrograde tracing; gastrocnemius muscle; spinal misdirection; sciatic nerve; peripheral nerve
10.  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.
doi:10.1097/NEN.0b013e31817e23f4
PMCID: PMC2748883  PMID: 18596543
Diabetes; Nerve regeneration; Prosaposin; Prosaptide TX14(A)
11.  Mechanisms of Enhancement of Neurite Regeneration In Vitro Following a Conditioning Sciatic Nerve Lesion 
To examine the mechanisms responsible for the more rapid nerve regeneration observed after a previous (conditioning) nerve injury, adult rats were subjected to a midthigh sciatic nerve transection by using one of three protocols designed to facilitate or restrict nerve regeneration: 1) ligation, in which transected axons were prevented from regenerating; 2) cut, in which transected axons were permitted to extend into peripheral target tissue but were separated from the denervated peripheral nerve stump; and 3) crush, in which axons could regenerate normally through the denervated distal nerve tract. The affected dorsal root ganglia (DRG) were subsequently removed, dissociated, and cultured for up to 3 days, and the timing of neurite initiation, rate of outgrowth, and arborization pattern of previously injured neurons were compared with control DRG. Our results indicate that conditioning lesions have at least four distinct and differentially regulated effects on neuronal morphogenesis: 1) conditioning lesions promote earlier neurite initiation, 2) prior nerve injury decreases the ability of neurons to extend long neurites following a second axotomy, 3) exposure to the environment of a denervated peripheral nerve stimulates greater initial rates of neurite outgrowth, and 4) conditioning lesions reduces initial neuritic branching frequency, resulting in straighter neurites whose growth cones extend further distances from their cell bodies. The primary effect of all conditioning lesions on cultured DRG neurons appeared to be to advance the timing of morphogenesis, resulting in conditioning-lesioned neurons that exhibited characteristics consistent with control neurons that had been cultured for an additional day or more. A secondary effect of conditioning lesions on neurite outgrowth rates was dependent on the local environment of the axons prior to culturing.
PMCID: PMC2605358  PMID: 9527536
neurite outgrowth; neurite initiation; nerve regeneration; conditioning lesion; neurite arborization
12.  Inhibition of calpains fails to improve regeneration through a peripheral nerve conduit 
Neuroscience Letters  2014;566(100):280-285.
Highlights
•Calpain inhibitor leupeptin locally applied to transected sciatic nerve of rats.•Axon number and myelination not significantly increased 3 months after lesion.•No difference in behavioral tests after nerve regeneration.
Intramuscular injection of the calpain inhibitor leupeptin promotes peripheral nerve regeneration in primates (Badalamente et al., 1989 [13]), and direct positive effects of leupeptin on axon outgrowth were observed in vitro (Hausott et al., 2012 [12]). In this study, we applied leupeptin (2 mg/ml) directly to collagen-filled nerve conduits in the rat sciatic nerve transection model. Analysis of myelinated axons and retrogradely labeled motoneurons as well as functional ‘CatWalk’ video analysis did not reveal significant differences between vehicle controls and leupeptin treated animals. Therefore, leupeptin does not improve nerve regeneration via protease inhibition in regrowing axons or in surrounding Schwann cells following a single application to a peripheral nerve conduit suggesting indirect effects on motor endplate integrity if applied systemically.
doi:10.1016/j.neulet.2014.03.009
PMCID: PMC4000267  PMID: 24631569
Leupeptin; Nerve lesion; Axotomy; Sciatic nerve; Peripheral nervous system
13.  Paeoniae alba Radix Promotes Peripheral Nerve Regeneration 
The present study provides in vitro and in vivo evaluation of Paeoniae alba Radix (PR) on peripheral nerve regeneration. In the in vitro study, we found the PR caused a marked enhancement of the nerve growth factor-mediated neurite outgrowth from PC12 cells as well as their expression of growth associated protein 43 and synapsin I. In the in vivo study, silicone rubber chambers filled with the PR water extract were used to bridge a 10-mm sciatic nerve defect in rats. At the conclusion of 8 weeks, regenerated nerves in the PR groups, especially at 1.25 mg ml−1 had a higher rate of successful regeneration across the wide gap, relatively larger mean values of total nerve area, myelinated axon count and blood vessel number, and a significantly larger nerve conductive velocity compared to the control group (P  <  .05). These results suggest that the PR extract can be a potential nerve growth-promoting factor, being salutary in aiding the growth of injured peripheral nerve.
doi:10.1093/ecam/nep115
PMCID: PMC3094696  PMID: 19687191
14.  EFFECT OF LOCALLY DELIVERED IGF-1 ON NERVE REGENERATION DURING AGING: AN EXPERIMENTAL STUDY IN RATS 
Muscle & nerve  2010;41(3):335-341.
Age is an important predictor of neuromuscular recovery after peripheral nerve injury. Insulin-like growth factor 1 (IGF-1) is a potent neurotrophic factor that is known to decline with increasing age. The purpose of this study was to determine if locally delivered IGF-1 would improve nerve regeneration and neuromuscular recovery in aged animals. Young and aged rats underwent nerve transection and repair with either saline or IGF-1 continuously delivered to the site of the nerve repair. After 3 months, nerve regeneration and neuromuscular junction morphology were assessed. In both young and aged animals, IGF-1 significantly improved axon number, diameter, and density. IGF-1 also significantly increased myelination and Schwann cell activity and preserved the morphology of the postsynaptic neuromuscular junction (NMJ). These results show that aged regenerating nerve is sensitive to IGF-1 treatment.
doi:10.1002/mus.21485
PMCID: PMC3045758  PMID: 19802878
aging; IGF-1; nerve injury; nerve regeneration; neuromuscular junction
15.  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.
Purpose.
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.
Methods.
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.
Results.
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).
Conclusions.
Although corneal NFD recovers to normal levels by 8 weeks after nerve transection, the arrangement of regenerated nerves is abnormal.
doi:10.1167/iovs.11-8332
PMCID: PMC3207999  PMID: 21896845
16.  Ultrastructural Changes in Spinal Motoneurons and Locomotor Functional Study after Sciatic Nerve Repair in Conduit Tube 
Objective(s)
Motor deficit and neuron degeneration is seen after nerve transection. The aim of this study is to determine whether a poled polyvinelidene fluoride (PVDF) tube with other supportive strategies can protect the neuronal morphology and motor function after sciatic nerve transaction in rats.
Materials and Methods
After transection of the left sciatic nerve in 60 male Wistar rats (200-250 g), the epineural group was sutured end to end. In the autograft rats, a 10 mm piece of sciatic nerve was rotated 180 °C and sutured back into the nerve gap. In the nerve guidance channel (NGC) group, polarized piezoelectric PVDF tube containing NGF and collagen gel was sutured in the gap. In control group sciatic nerve was removed (10 mm) without repair. After one, four and eight weeks, the L4-L6 spinal cord segment was removed for histological study using transmission electron microscope. Functional outcome was assessed using the Basso, Bresnahan and Beattie (BBB) locomotor scale at both four and eight weeks after the lesion.
Results
Chromatin condensation was seen after 4 weeks in the repair groups. Cell membrane shrinkage and mitochondrial degeneration was observed after 4 and 8 weeks respectively, in the autografted and NGC rats. In the control group, chromatin condensation, cell membrane shrinkage with mitochondrial degeneration and vacuolization of perikaryon was seen after 1, 4 and 8 weeks, respectively. At 56 days, the functional recovery of the epineural rats significantly increased in comparison to the other groups (P< 0.05).
Conclusion
The epineural suture has more efficacies, and NGC may be used as a proper substitute for autograft in nerve injury.
PMCID: PMC3586910  PMID: 23492837
Motor neuron; Rat; Recovery; Sciatic nerve
17.  Binary Imaging Analysis for Comprehensive Quantitative Assessment of Peripheral Nerve 
Journal of neuroscience methods  2007;166(1):116-124.
Quantitative histomorphometry is the current gold standard for objective measurement of nerve architecture and its components. Many methods still in use rely heavily upon manual techniques that are prohibitively time consuming, predisposing to operator fatigue, sampling error, and overall limited reproducibility. More recently, investigators have attempted to combine the speed of automated morphometry with the accuracy of manual and semi-automated methods. Systematic refinements in binary imaging analysis techniques combined with an algorithmic approach allow for more exhaustive characterization of nerve parameters in the surgically relevant injury paradigms of regeneration following crush, transection, and nerve gap injuries. The binary imaging method introduced here uses multiple bitplanes to achieve reproducible, high throughput quantitative assessment of peripheral nerve. Number of myelinated axons, myelinated fiber diameter, myelin thickness, fiber distributions, myelinated fiber density, and neural debris can be quantitatively evaluated with stratification of raw data by nerve component. Results of this semi-automated method are validated by comparing values against those obtained with manual techniques. The use of this approach results in more rapid, accurate, and complete assessment of myelinated axons than manual techniques.
doi:10.1016/j.jneumeth.2007.06.018
PMCID: PMC2587177  PMID: 17675163
18.  Lack of effectiveness of laser therapy applied to the nerve course and the correspondent medullary roots 
Acta Ortopedica Brasileira  2013;21(2):92-97.
OBJECTIVE:
To investigate the influence of low intensity laser irradiation on the regeneration of the fibular nerve of rats after crush injury.
METHODS:
Twenty-five rats were used, divided into three groups: 1) intact nerve, no treatment; 2) crushed nerve, no treatment; 3) crush injury, laser irradiation applied on the medullary region corresponding to the roots of the sciatic nerve and subsequently on the course of the damaged nerve. Laser irradiation was carried out for 14 consecutive days.
RESULTS:
Animals were evaluated by functional gait analysis with the peroneal functional index and by histomorphometric analysis using the total number of myelinated nerve fibers and their density, total number of Schwann cells, total number of blood vessels and the occupied area, minimum diameter of the fiber diameter and G-quotient.
CONCLUSION:
According to the statistical analysis there was no significant difference among groups and the authors conclude that low intensity laser irradiation has little or no influence on nerve regeneration and functional recovery. Laboratory investigation.
doi:10.1590/S1413-78522013000200005
PMCID: PMC3861963  PMID: 24453650
Nerve regeneration; Rats; Crush injury; Fibular nerve; Laser therapy
19.  Swimming Exercise in the Acute or Late Phase after Sciatic Nerve Crush Accelerates Nerve Regeneration 
Neural Plasticity  2011;2011:783901.
There is no consensus about the best time to start exercise after peripheral nerve injury. We evaluated the morphological and functional characteristics of the sciatic nerves of rats that began to swim immediately after crush nerve injury (CS1), those that began to swim 14 days after injury (CS14), injured rats not submitted to swimming (C), and uninjured rats submitted to swimming (S). After 30 days the number of axons in CS1 and CS14 was lower than in C (P < 0.01). The diameter of axons and nerve fibers was larger in CS1 (P < 0.01) and CS14 (P < 0.05) than in C, and myelin sheath thickness was lower in all crushed groups (P < 0.05). There was no functional difference between CS1 and CS14 (P > 0.05). Swimming exercise applied during the acute or late phase of nerve injury accelerated nerve regeneration and synaptic elimination after axonotmesis, suggesting that exercise may be initiated immediately after injury.
doi:10.1155/2011/783901
PMCID: PMC3159303  PMID: 21876821
20.  The value of an operating microscope in peripheral nerve repair  
International Orthopaedics  1998;22(2):107-110.
Summary.
The aim of this study was to test the hypothesis that the use of an operating microscope improves the results of peripheral nerve repair. Tibial nerve grafting was carried out on 48 Fischer rats divided into 2 groups: in one, a loupe was used, and in the other a surgical microscope. At 5 months after grafting, recovery was evaluated by functional, electromyographic, and morphometric tests. The mean motor nerve conduction velocity was 26.77±9.37 m/sec in the group where the loupe was used compared with 44.19±11.36 m/s when the microscope group was used. The soleus muscle weight and the diameter of myelinated fibres also confirmed better regeneration in the microscope group. These results clearly indicate that it is essential to use the microscope for peripheral nerve repair.
doi:10.1007/s002640050219
PMCID: PMC3619702  PMID: 9651776
21.  Preliminary Investigation on Use of High-Resolution Optical Coherence Tomography to Monitor Injury and Repair in the Rat Sciatic Nerve 
Lasers in surgery and medicine  2010;42(4):306-312.
Background and Objective
Optical coherence tomography (OCT) has been used in limited settings to study peripheral nerve injury. The purpose of the study is to determine whether high-resolution OCT can be used to monitor nerve injury and regeneration in the rat sciatic nerve following crush injury, ligation, and transection with microsurgical repair.
Study Design/Materials and Methods
Forty-five rats were segregated into three groups. The right sciatic nerve was suture ligated (n = 15), cut then microsurgically repaired (n = 15), or crushed (n = 15). The left sciatic nerve served as the control; only surgical exposure and skin closure were performed. Each group was further divided into three subgroups where they were assigned survival durations of 4, 15, or 24 weeks. Following euthanasia, nerves were harvested, fixed in formalin, and imaged at the injury site, as well as proximal and distal ends. The OCT system resolution was approximately 7 μm in tissue with a 1,060 nm central wavelength.
Results
Control (uninjured) nerve tissue showed homogenous signal distribution to a relatively uniform depth; in contrast, damaged nerves showed irregular signal distribution and intensity. Changes in signal distribution were most significant at the injury site and distal regions. Increases in signal irregularity were evident during longer recovery times. Histological analysis determined that OCT imaging was limited to the surrounding perineurium and scar tissue.
Conclusion
OCT has the potential to be a valuable tool for monitoring nerve injury and repair, and the changes that accompany wound healing, providing clinicians with a non-invasive tool to treat nerve injuries.
doi:10.1002/lsm.20915
PMCID: PMC2898724  PMID: 20432279
nerve injury; peripheral nerve; optical coherence tomography and nerve
22.  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
23.  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.
doi:10.2119/molmed.2012.00248
PMCID: PMC3646097  PMID: 23508572
24.  ATF3 upregulation in glia during Wallerian degeneration: differential expression in peripheral nerves and CNS white matter 
BMC Neuroscience  2004;5:9.
Background
Many changes in gene expression occur in distal stumps of injured nerves but the transcriptional control of these events is poorly understood. We have examined the expression of the transcription factors ATF3 and c-Jun by non-neuronal cells during Wallerian degeneration following injury to sciatic nerves, dorsal roots and optic nerves of rats and mice, using immunohistochemistry and in situ hybridization.
Results
Following sciatic nerve injury – transection or transection and reanastomosis – ATF3 was strongly upregulated by endoneurial, but not perineurial cells, of the distal stumps of the nerves by 1 day post operation (dpo) and remained strongly expressed in the endoneurium at 30 dpo when axonal regeneration was prevented. Most ATF3+ cells were immunoreactive for the Schwann cell marker, S100. When the nerve was transected and reanastomosed, allowing regeneration of axons, most ATF3 expression had been downregulated by 30 dpo. ATF3 expression was weaker in the proximal stumps of the injured nerves than in the distal stumps and present in fewer cells at all times after injury. ATF3 was upregulated by endoneurial cells in the distal stumps of injured neonatal rat sciatic nerves, but more weakly than in adult animals. ATF3 expression in transected sciatic nerves of mice was similar to that in rats. Following dorsal root injury in adult rats, ATF3 was upregulated in the part of the root between the lesion and the spinal cord (containing Schwann cells), beginning at 1 dpo, but not in the dorsal root entry zone or in the degenerating dorsal column of the spinal cord. Following optic nerve crush in adult rats, ATF3 was found in some cells at the injury site and small numbers of cells within the optic nerve displayed weak immunoreactivity. The pattern of expression of c-Jun in all types of nerve injury was similar to that of ATF3.
Conclusion
These findings raise the possibility that ATF3/c-Jun heterodimers may play a role in regulating changes in gene expression necessary for preparing the distal segments of injured peripheral nerves for axonal regeneration. The absence of the ATF3 and c-Jun from CNS glia during Wallerian degeneration may limit their ability to support regeneration.
doi:10.1186/1471-2202-5-9
PMCID: PMC400733  PMID: 15113454
25.  Misdirection of Regenerating Axons and Functional Recovery Following Sciatic Nerve Injury in Rats 
Poor functional recovery found after peripheral nerve injury has been attributed to the misdirection of regenerating axons to reinnervate functionally inappropriate muscles. We applied brief electrical stimulation (ES) to the common fibular (CF) but not the tibial (Tib) nerve just prior to transection and repair of the entire rat sciatic nerve, to attempt to influence the misdirection of its regenerating axons. The specificity with which regenerating axons reinnervated appropriate targets was evaluated physiologically using compound muscle action potentials (M responses) evoked from stimulation of the two nerve branches above the injury site. Functional recovery was assayed using the timing of electromyography (EMG) activity recorded from the tibialis anterior (TA) and soleus (Sol) muscles during treadmill locomotion and kinematic analysis of hindlimb locomotor movements. Selective ES of the CF nerve resulted in restored M-responses at earlier times than in unstimulated controls in both TA and Sol muscles. Stimulated CF axons reinnervated inappropriate targets to a greater extent than unstimulated Tib axons. During locomotion, functional antagonist muscles, TA and Sol, were coactivated both in stimulated rats and in unstimulated but injured rats. Hindlimb kinematics in stimulated rats were comparable to untreated rats, but significantly different from intact controls. Selective ES promotes enhanced axon regeneration but does so with decreased fidelity of muscle reinnervation. Functional recovery is neither improved nor degraded, suggesting that compensatory changes in the outputs of the spinal circuits driving locomotion may occur irrespective of the extent of misdirection of regenerating axons in the periphery.
doi:10.1002/cne.22446
PMCID: PMC3703664  PMID: 21120925
peripheral nerve; regeneration; electrical stimulation; neuromuscular specificity; electromyography; locomotion

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