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1.  Effects of Treatment of Treadmill Combined with Electro-Acupuncture on Tibia Bone Mass and Substance PExpression of Rabbits with Sciatic Nerve Injury 
PLoS ONE  2016;11(11):e0164652.
The peripheral nervous system may play an important role in normal bone maintenance and remodeling. Substance P (SP) is a neuropeptide associated with bone loss and formation that may mediate the effects of the nervous system. The purpose of this study is to determine if treadmill running combined with electro-acupuncture at Jiaji acupoints (Jiaji-EA) affects tibial bone mass and SP expression in rabbits with sciatic nerve injury. Twenty-four juvenile male New Zealand white rabbits were randomly assigned to one of 4 groups: sham injury control (sham), sciatic never crush control (SNCr), treadmill running (treadmill), and Jiaji-EA combined with treadmill running (ET group). The SNCr, treadmill, and ET groups all had an induced sciatic never crush injury of approximately 2mm. Control groups received no intervention; the treadmill and ET groups were trained by treadmill; the ET group also received Jiaji-EA. After the 4 weeks of treatment, toe-spreading index (TSI), BMD, bone strength, and SP expression in the tibia were significantly lower in the nerve injury groups (SNCr, treadmill, and ET) compared to the sham groups (p<0.05). Treatment (treadmill and ET groups) increased all measures compared to the SNCr group (p<0.05). Further, TSI, BMD, bone strength, and SP expression in the ET group were higher than the treadmill group (p<0.05). Our results indicate that treadmill therapy combined with electro-acupuncture at Jiaji acupoints prevents bone loss in rabbit tibias after sciatic nerve injury. This may occur in two ways: indirectly in association with axon regeneration and directly via loading on the bone mediated through increased SP expression. This study provides important evidence for the clinical treatment of bone loss after peripheral nerve injury.
doi:10.1371/journal.pone.0164652
PMCID: PMC5120789  PMID: 27880769
2.  Impairment of toll-like receptors 2 and 4 leads to compensatory mechanisms after sciatic nerve axotomy 
Background
Peripheral nerve injury results in retrograde cell body-related changes in the spinal motoneurons that will contribute to the regenerative response of their axons. Successful functional recovery also depends on molecular events mediated by innate immune response during Wallerian degeneration in the nerve microenvironment. A previous study in our lab demonstrated that TLR 2 and 4 develop opposite effects on synaptic stability in the spinal cord after peripheral nerve injury. Therefore, we suggested that the better preservation of spinal cord microenvironment would positively influence distal axonal regrowth. In this context, the present work aimed to investigate the influence of TLR2 and TLR4 on regeneration and functional recovery after peripheral nerve injury.
Methods
Eighty-eight mice were anesthetized and subjected to unilateral sciatic nerve crush (C3H/HeJ, n = 22, C3H/HePas, n = 22; C57Bl6/J, n = 22 and TLR2−/−, n = 22). After the appropriate survival times (3, 7, 14 days, and 5 weeks), all mice were killed and the sciatic nerves and tibialis cranialis muscles were processed for immunohistochemistry and transmission electron microscopy (TEM). Gait analysis, after sciatic nerve crushing, was performed in another set of mice (minimum of n = 8 per group), by using the walking track test (CatWalk system).
Results
TLR4 mutant mice presented greater functional recovery as well as an enhanced p75NTR and neurofilament protein expression as compared to the wild-type strain. Moreover, the better functional recovery in mutant mice was correlated to a greater number of nerve terminal sprouts. Knockout mice for TLR2 exhibited 30 % greater number of degenerated axons in the distal stump of the sciatic nerve and a decreased p75NTR and neurofilament protein expression compared to the wild type. However, the absence of TLR2 receptor did not influence the overall functional recovery. End-point equivalent functional recovery in transgenic mice may be a result of enhanced axonal diameter found at 2 weeks after lesion.
Conclusions
Altogether, the present results indicate that the lack of TLR2 or the absence of functional TLR4 does affect the nerve regeneration process; however, such changes are minimized through different compensatory mechanisms, resulting in similar motor function recovery, as compared to wild-type mice. These findings contribute to the concept that innate immune-related molecules influence peripheral nerve regeneration by concurrently participating in processes taking place both at the CNS and PNS.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-016-0579-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12974-016-0579-6
PMCID: PMC4879730  PMID: 27222120
Pheripheral nerve injury; Axotomy; Toll-like receptors and functional recovery
3.  A protocol of histone modification-based mechanistic study of acupuncture in patients with stable angina pectoris 
Background
Angina pectoris (Angina) is a medical condition related to myocardial ischemia. Although acupuncture has been widely accepted as a clinical approach for angina, there is no sufficient evidence of its effectiveness against this syndrome, and its mechanisms have not yet been well elucidated. We develop this protocol to confirm the clinical efficacy of electro-acupuncture on stable angina pectoris by needling on acupoint Neiguan (PC6). Furthermore, we employ high-throughput sequencing technology to investigate the gene expression profiling and determine involvement of histone modifications in the regulation of genes after electro-acupuncture treatment.
Methods/Design
A randomized, controlled, double-blinded (assessor and patients) trial will be carried out. Sixty participants will be randomly assigned to two acupuncture treatment groups and one control group in a 1:1:1 ratio. Participants in acupuncture groups will receive 12 sessions of electro-acupuncture treatment across 4 weeks, followed by a 12-week randomization period. The acupuncture groups are divided into Neiguan (PC6) on Pericardium Meridian of Hand-jueyin or a non-acupoint. The primary clinical measure of effect is the frequency of angina attacks between these groups for four weeks after randomization. RNAs are extracted from peripheral neutrophils collected from all participants on day 0, day 30, and week 16, and are processed to RNA-Seq. We then investigate profiles of histone modifications by ChIP-Seq, for H3 Lysine 4 (H3K4me) and acetylation of H3 Lysine 27 (H3K27ac), in the presence or absence of acupuncture treatment.
Discussion
This study determines the efficacy and mechanisms of electro-acupuncture on stable angina pectoris. We focus on effectiveness of acupuncture on alleviating symptoms of myocardial ischemia and the gene regulation and the chromatin remodeling marks, including H3K4me1, H3K4me2, and H3K27ac, which could be key factors for regulating gene expressions caused by electro-acupuncture treatment at Neiguan. This is the first genome-wide study of electro-acupuncture treatment in angina patients, and will provide valuable information for future studies in the fields of acupuncture and its underlying mechanisms.
Fourteen patients have been recruited since recruitment opened in November of 2012. This study is scheduled to end in November of 2014.
Trials registration
ChiCTR-TRC-12002668
doi:10.1186/s12906-015-0653-0
PMCID: PMC4465012  PMID: 25925670
Acupuncture; Stable angina pectoris; Study protocol; Gene profiling; Histone modification
4.  Cytidine 5′-diphosphocholine administration prevents peripheral neuropathic pain after sciatic nerve crush injury in rats 
Journal of Pain Research  2016;9:287-291.
Background
Cytidine 5′-diphosphocholine (citicoline) has been shown to have beneficial effects in central nervous system injury as well as in motoric functional recovery after peripheral nerve injury. This study aimed to examine the effect of citicoline on prevention of neuropathic pain in a rat model of sciatic nerve crush injury.
Methods
Forty experimental rats were divided into four groups. In three groups, the right sciatic nerves were crushed in the mid-thigh region, and a gelatin sponge moistened with 0.4 or 0.8 mL of 100 µmol/L citicoline, or saline 0.4 mL in the control group, was applied. The fourth group of rats was sham-operated, ie the sciatic nerve was exposed with no crush. Functional assessments were performed 4 weeks after crush injury. von Frey filaments (100 g threshold) were used to assess neuropathic pain. In addition, the sciatic functional index and extensor postural thrust (EPT) tests were used to assess motoric function.
Results
The crush/citicoline 0.4 mL group had a lower percentage of pain (23.53%, n=17) compared with the crush/saline group (53.33%, n=15, P<0.005). The crush/citicoline 0.4 mL group also showed better motoric recovery, as seen in stronger EPT results (P<0.001). However, the sciatic functional index analysis did not show significant differences between groups (P=0.35). The crush/citicoline 0.8 mL group showed a higher percentage of pain (66.67%, n=18) and less EPT recovery. These results may be explained by more severe nerve injury due to compression with a larger administered volume.
Conclusion
In situ administration of 0.4 mL of 100 µmol/L citicoline prevents the occurrence of neuropathic pain and induces motoric recovery, evaluated by EPT test, 4 weeks after sciatic nerve injury.
doi:10.2147/JPR.S70481
PMCID: PMC4883808  PMID: 27284264
nerve injury; nerve regeneration; neuropathic pain
5.  Reinnervation of the Tibialis Anterior Following Sciatic Nerve Crush Injury: A Confocal Microscopic Study in Transgenic Mice 
Experimental neurology  2007;207(1):64-74.
Transgenic mice whose axons and Schwann cells express fluorescent chromophores enable new imaging techniques and augment concepts in developmental neurobiology. The utility of these tools in the study of traumatic nerve injury depends on employing nerve models that are amenable to microsurgical manipulation and gauging functional recovery. Motor recovery from sciatic nerve crush injury is studied here by evaluating motor endplates of the tibialis anterior muscle, which is innervated by the deep peroneal branch of the sciatic nerve. Following sciatic nerve crush, the deep surface of the tibialis anterior muscle is examined using whole mount confocal microscopy, and reinnervation is characterized by imaging fluorescent axons or Schwann cells (SCs). One week following sciatic crush injury, 100% of motor endplates are denervated with partial reinnervation at two weeks, hyperinnervation at three and four weeks, and restoration of a 1:1 axon to motor endplate relationship six weeks after injury. Walking track analysis reveals progressive recovery of sciatic nerve function by six weeks. SCs reveal reduced S100 expression within two weeks of denervation, correlating with regression to a more immature phenotype. Reinnervation of SCs restores S100 expression and a fully differentiated phenotype. Following denervation, there is altered morphology of circumscribed terminal Schwann cells demonstrating extensive process formation between adjacent motor endplates. The thin, uniformly innervated tibialis anterior muscle is well suited for studying motor reinnervation following sciatic nerve injury. Confocal microscopy may be performed coincident with other techniques of assessing nerve regeneration and functional recovery.
doi:10.1016/j.expneurol.2007.05.028
PMCID: PMC2000860  PMID: 17628540
transgenic mice; chromophore; sciatic nerve crush; motor endplate
6.  Deficiency in Monocarboxylate Transporter 1 (MCT1) in Mice Delays Regeneration of Peripheral Nerves following Sciatic Nerve Crush 
Experimental neurology  2014;263:325-338.
Peripheral nerve regeneration following injury occurs spontaneously, but many of the processes require metabolic energy. The mechanism of energy supply to axons has not previously been determined. In the central nervous system, monocarboxylate transporter 1 (MCT1), expressed in oligodendroglia, is critical for supplying lactate or other energy metabolites to axons. In the current study, MCT1 is shown to localize within the peripheral nervous system to perineurial cells, dorsal root ganglion neurons, and Schwann cells by MCT1 immunofluorescence and MCT1 tdTomato BAC reporter mice. To investigate whether MCT1 is necessary for peripheral nerve regeneration, sciatic nerves in MCT1 heterozygous null mice are crushed and peripheral nerve regeneration quantified electrophysiologically and anatomically. Compound muscle action potential (CMAP) recovery is delayed from a median of 21 days in wild-type mice to greater than 38 days in MCT1 heterozygote null mice. In fact, half of the MCT1 heterozygote null mice have no recovery of CMAP at 42 days, while all of the wild-type mice recovered. In addition, muscle fibers remain 40% more atrophic and neuromuscular junctions 40% more denervated at 42 days post-crush in the MCT1 heterozygote null mice than wild-type mice. The delay in nerve regeneration is not only in motor axons, as the number of regenerated axons in the sural sensory nerve of MCT1 heterozygote null mice at 4 weeks and tibial mixed sensory and motor nerve at 3 weeks is also significantly reduced compared to wild-type mice. This delay in regeneration may be partly through failed Schwann cell function, as there is reduced early phagocytosis of myelin debris and remyelination of axon segments. These data for the first time demonstrate that MCT1 is critical for regeneration of both sensory and motor axons in mice following sciatic nerve crush.
doi:10.1016/j.expneurol.2014.10.018
PMCID: PMC4292924  PMID: 25447940
monocarboxylate transporter; metabolism; regeneration; peripheral nerve; nerve crush; perineurial cell; Schwann cell; dorsal root ganglion; axon; electron microscopy
7.  Acupuncture for treating fibromyalgia 
Background
One in five fibromyalgia sufferers use acupuncture treatment within two years of diagnosis.
Objectives
To examine the benefits and safety of acupuncture treatment for fibromyalgia.
Search methods
We searched CENTRAL, PubMed, EMBASE, CINAHL, National Research Register, HSR Project and Current Contents, as well as the Chinese databases VIP and Wangfang to January 2012 with no language restrictions.
Selection criteria
Randomised and quasi-randomised studies evaluating any type of invasive acupuncture for fibromyalgia diagnosed according to the American College of Rheumatology (ACR) criteria, and reporting any main outcome: pain, physical function, fatigue, sleep, total well-being, stiffness and adverse events.
Data collection and analysis
Two author pairs selected trials, extracted data and assessed risk of bias. Treatment effects were reported as standardised mean differences (SMD) and 95%confidence intervals (CI) for continuous outcomes using different measurement tools (pain, physical function, fatigue, sleep, total well-being and stiffness) and risk ratio (RR) and 95% CI for dichotomous outcomes (adverse events).We pooled data using the random-effects model.
Main results
Nine trials (395 participants) were included. All studies except one were at low risk of selection bias; five were at risk of selective reporting bias (favouring either treatment group); two were subject to attrition bias (favouring acupuncture); three were subject to performance bias (favouring acupuncture) and one to detection bias (favouring acupuncture). Three studies utilised electro-acupuncture (EA) with the remainder using manual acupuncture (MA) without electrical stimulation. All studies used ’formula acupuncture’ except for one, which used trigger points.
Low quality evidence from one study (13 participants) showed EA improved symptoms with no adverse events at one month following treatment. Mean pain in the non-treatment control group was 70 points on a 100 point scale; EA reduced pain by a mean of 22 points (95% confidence interval (CI) 4 to 41), or 22% absolute improvement. Control group global well-being was 66.5 points on a 100 point scale; EA improved well-being by a mean of 15 points (95% CI 5 to 26 points). Control group stiffness was 4.8 points on a 0 to 10 point; EA reduced stiffness by a mean of 0.9 points (95% CI 0.1 to 2 points; absolute reduction 9%, 95% CI 4% to 16%). Fatigue was 4.5 points (10 point scale) without treatment; EA reduced fatigue by a mean of 1 point (95% CI 0.22 to 2 points), absolute reduction 11% (2% to 20%). There was no difference in sleep quality (MD 0.4 points, 95% CI −1 to 0.21 points, 10 point scale), and physical function was not reported.
Moderate quality evidence from six studies (286 participants) indicated that acupuncture (EA or MA) was no better than sham acupuncture, except for less stiffness at one month. Subgroup analysis of two studies (104 participants) indicated benefits of EA. Mean pain was 70 points on 0 to 100 point scale with sham treatment; EA reduced pain by 13% (5% to 22%); (SMD −0.63, 95% CI −1.02 to −0.23). Global well-being was 5.2 points on a 10 point scale with sham treatment; EA improved well-being: SMD 0.65, 95% CI 0.26 to 1.05; absolute improvement 11% (4% to 17%). EA improved sleep, from 3 points on a 0 to 10 point scale in the sham group: SMD 0.40 (95% CI 0.01 to 0.79); absolute improvement 8% (0.2% to 16%). Low-quality evidence from one study suggested that MA group resulted in poorer physical function: mean function in the sham group was 28 points (100 point scale); treatment worsened function by a mean of 6 points (95% CI −10.9 to −0.7). Low-quality evidence from three trials (289 participants) suggested no difference in adverse events between real (9%) and sham acupuncture (35%); RR 0.44 (95% CI 0.12 to 1.63).
Moderate quality evidence from one study (58 participants) found that compared with standard therapy alone (antidepressants and exercise), adjunct acupuncture therapy reduced pain at one month after treatment: mean pain was 8 points on a 0 to 10 point scale in the standard therapy group; treatment reduced pain by 3 points (95% CI −3.9 to −2.1), an absolute reduction of 30% (21% to 39%). Two people treated with acupuncture reported adverse events; there were none in the control group (RR 3.57; 95% CI 0.18 to 71.21). Global well-being, sleep, fatigue and stiffness were not reported. Physical function data were not usable.
Low quality evidence from one study (38 participants) showed a short-term benefit of acupuncture over antidepressants in pain relief: mean pain was 29 points (0 to 100 point scale) in the antidepressant group; acupuncture reduced pain by 17 points (95% CI −24.1 to −10.5). Other outcomes or adverse events were not reported.
Moderate-quality evidence from one study (41 participants) indicated that deep needling with or without deqi did not differ in pain, fatigue, function or adverse events. Other outcomes were not reported.
Four studies reported no differences between acupuncture and control or other treatments described at six to seven months follow-up.
No serious adverse events were reported, but there were insufficient adverse events to be certain of the risks.
Authors’ conclusions
There is low tomoderate-level evidence that compared with no treatment and standard therapy, acupuncture improves pain and stiffness in people with fibromyalgia. There is moderate-level evidence that the effect of acupuncture does not differ from sham acupuncture in reducing pain or fatigue, or improving sleep or global well-being. EA is probably better than MA for pain and stiffness reduction and improvement of global well-being, sleep and fatigue. The effect lasts up to one month, but is not maintained at six months follow-up. MA probably does not improve pain or physical functioning. Acupuncture appears safe. People with fibromyalgia may consider using EA alone or with exercise and medication. The small sample size, scarcity of studies for each comparison, lack of an ideal sham acupuncture weaken the level of evidence and its clinical implications. Larger studies are warranted.
doi:10.1002/14651858.CD007070.pub2
PMCID: PMC4105202  PMID: 23728665
Acupuncture Therapy [*methods]; Fibromyalgia [*therapy]; Pain Management [methods]; Randomized Controlled Trials as Topic; Humans
8.  Electro-acupuncture promotes survival, differentiation of the bone marrow mesenchymal stem cells as well as functional recovery in the spinal cord-transected rats 
BMC Neuroscience  2009;10:35.
Background
Bone marrow mesenchymal stem cells (MSCs) are one of the potential tools for treatment of the spinal cord injury; however, the survival and differentiation of MSCs in an injured spinal cord still need to be improved. In the present study, we investigated whether Governor Vessel electro-acupuncture (EA) could efficiently promote bone marrow mesenchymal stem cells (MSCs) survival and differentiation, axonal regeneration and finally, functional recovery in the transected spinal cord.
Results
The spinal cords of adult Sprague-Dawley (SD) rats were completely transected at T10, five experimental groups were performed: 1. sham operated control (Sham-control); 2. operated control (Op-control); 3. electro-acupuncture treatment (EA); 4. MSCs transplantation (MSCs); and 5. MSCs transplantation combined with electro-acupuncture (MSCs+EA). After 2-8 weeks of MSCs transplantation plus EA treatment, we found that the neurotrophin-3 (NT-3), cAMP level, the differentiation of MSCs, the 5-HT positive and CGRP positive nerve fibers in the lesion site and nearby tissue of injured spinal cord were significantly increased in the MSCs+EA group as compared to the group of the MSCs transplantation or the EA treated alone. Furthermore, behavioral test and spinal cord evoked potentials detection demonstrated a significantly functional recovery in the MSCs +EA group.
Conclusion
These results suggest that EA treatment may promote grafted MSCs survival and differentiation; MSCs transplantation combined with EA treatment could promote axonal regeneration and partial locomotor functional recovery in the transected spinal cord in rats and indicate a promising avenue of treatment of spinal cord injury.
doi:10.1186/1471-2202-10-35
PMCID: PMC2679038  PMID: 19374777
9.  Reduced Renshaw Recurrent Inhibition after Neonatal Sciatic Nerve Crush in Rats 
Neural Plasticity  2014;2014:786985.
Renshaw recurrent inhibition (RI) plays an important gated role in spinal motion circuit. Peripheral nerve injury is a common disease in clinic. Our current research was designed to investigate the change of the recurrent inhibitory function in the spinal cord after the peripheral nerve crush injury in neonatal rat. Sciatic nerve crush was performed on 5-day-old rat puppies and the recurrent inhibition between lateral gastrocnemius-soleus (LG-S) and medial gastrocnemius (MG) motor pools was assessed by conditioning monosynaptic reflexes (MSR) elicited from the sectioned dorsal roots and recorded either from the LG-S and MG nerves by antidromic stimulation of the synergist muscle nerve. Our results demonstrated that the MSR recorded from both LG-S or MG nerves had larger amplitude and longer latency after neonatal sciatic nerve crush. The RI in both LG-S and MG motoneuron pools was significantly reduced to virtual loss (15–20% of the normal RI size) even after a long recovery period upto 30 weeks after nerve crush. Further, the degree of the RI reduction after tibial nerve crush was much less than that after sciatic nerve crush indicatig that the neuron-muscle disconnection time is vital to the recovery of the spinal neuronal circuit function during reinnervation. In addition, sciatic nerve crush injury did not cause any spinal motor neuron loss but severally damaged peripheral muscle structure and function. In conclusion, our results suggest that peripheral nerve injury during neonatal early development period would cause a more sever spinal cord inhibitory circuit damage, particularly to the Renshaw recurrent inhibition pathway, which might be the target of neuroregeneration therapy.
doi:10.1155/2014/786985
PMCID: PMC3981522  PMID: 24778886
10.  Traumatic neuroma in continuity injury model in rodents: a preliminary report 
Selected abstracts delivered at the 8th Annual AOSpine North America Fellows Forum
Consistent with EBSJ's commitment to fostering quality research, we are pleased to feature some of the most highly rated abstracts from the 8th Annual AOSpine North America Fellows Forum in Banff Canada. Enhancing the quality of evidence in spine care means acknowledging and supporting the efforts of young researchers within our AOSpine North America network. We look forward to seeing more from these promising researchers in the future.
Abstract
Study type: Basic science research report
Introduction: Spinal nerve-injury management and prevention constitute a substantial proportion of a spinal surgeon's practice. Functional recovery after peripheral nerve injuries is often unsatisfactory and to optimize the outcomes, an intimate understanding of these injuries is required. Sunderland classified peripheral nerve injuries into five grades.1 Grade 1 (neurapraxia) and grade 2 (axonal disruption) injuries usually recover with no or insignificant functional deficits within weeks to a few months, respectively. Injuries that are most difficult to manage clinically are the often mixed grade 3 (endoneurial disruption) and grade 4 (perineurial disruption) lesions where spontaneous functional recovery is limited or absent, resulting in neuroma in continuity (NIC). Traumatic NIC is characterized by aberrant intra- and extra- fascicular axonal regeneration and scar formation within an unsevered injured nerve, resulting in impaired and erroneous end-organ reinnervation.2,3 Animal models reproducing grade 1, 2, 3, and 5 lesions have been developed, but to our knowledge a clinically relevant rodent model of NIC has not been developed.4,5,6,7,8 The effective peripheral nerve regeneration and resilience of rodents make it challenging to recreate the NIC scenario.
Objective: Our goal was to develop a practical rodent model for focal traumatic NIC, demonstrating the characteristic histological features, supported by concordant functional deficits. Such a model may help us to identify this injury pattern earlier and allow development of intervention strategies to reduce neuronal misdirection, scar formation, and enhance regeneration for improved functional recovery.
Methods: Various injury techniques were tested on freshly harvested Lewis rat sciatic nerves ex vivo, and examined histologically before inflicting more refined injuries in vivo. The optimal experimental injuries combined a 50 g traction force applied with a spring scale hooked around the sciatic nerve, and focal three second maximal compression using a malleus nipper (Figure 1). Nerves were harvested at 0, 5, 13, 21, and 65 days, and processed for longitudinal 8 micron cryostat sectioning, H&E, laminin, neurofilament, and Masson's trichrome staining. Skilled locomotion (tapered beam, ladder rung) and flat plane locomotion for ground reaction force (GRF) analysis were performed serially up to 9 weeks with the experimental (n = 4) and simple (control) crush (n = 1) injuries by blinded animal behavior experts, using methods as recently described.9
Photograph illustrating the experimental injury. Fifty grams of traction is applied in a direction orthogonal to the native nerve course after external neurolysis, simultaneously, three second maximal compression is applied at the sciatic trifurcation, just distal to a mesoneurial suture. Malleus nipper with tip detail and 100 g spring scale in bottom left. In situ sciatic nerve immediately after injury (top right).
Results: Disruption of the endoneurium and perineurium with aberrant intra- and extrafascicular axonal regeneration and progressive fibrosis was consistently demonstrated histologically in ten out of ten nerves with experimental injuries. In contrast, crush injuries showed only signs of Wallerian degeneration (Figure 2). At 8 weeks, experimental animals made more errors during skilled locomotion as compared to nerve crush animals. GRFs revealed impaired vertical and fore-aft force generation by the injured limbs at week 9 in the experimental group, whereas GRFs from the simple crush animal revealed recovery at the same time point (Figure 3).
Injury zones at five days (a–d, bar = 200 µm) and 65 days (e–h, bar = 50 µm), comparing crush (top) to experimental (bottom) injuries; Masson's trichrome and neurofilament. Note the aberrant axonal sprouting and regeneration in the experimental injury group, associated with increased intrafascicular collagen, in contrast to orderly regeneration and lack of scar in the simple crush group.
Mean vertical and fore-aft ground reaction forces at both baseline and 9 weeks from representative animals. Compared to baseline and crush-injured animal at 9 weeks, animals in the experimental group bear less weight on both their right (surgical) hind limb (solid line), and fore limb (dotted line) at 9 weeks. Comparable with historical data, the crush animal have improved braking (*) and propulsive (#) forces in fore and hind limbs (injured side) compared to the experimental group, though these have not returned to baseline values.
Conclusions: We have demonstrated histological features and poor functional recovery consistent with NIC formation in a rodent model. The injury mechanism employed combines traction and compression forces akin to the physical forces at play in clinical nerve injuries. Additional validating experiments are in progress.
doi:10.1055/s-0028-1100915
PMCID: PMC3623102  PMID: 23637668
Locomotion; nerve regeneration; Sunderland grade 4 nerve injury
11.  Artemin Induced Functional Recovery And Reinnervation After Partial Nerve Injury 
Pain  2013;155(3):476-484.
Systemic artemin promotes regeneration of dorsal roots to the spinal cord following crush injury. However, it is unclear whether systemic artemin can promote peripheral nerve regeneration and functional recovery distal to the dorsal root ganglion (DRG). In the present investigation, male SD rats received axotomy, ligation, or crush of the L5 spinal nerve or sham surgery. Starting the day of injury, animals received intermittent s.c. artemin or vehicle across 2 weeks. Sensory thresholds to tactile or thermal stimuli were monitored for 6 weeks following injury. Immunohistochemical analyses of the DRG and nerve regeneration were performed at the 6 week timepoint. Artemin transiently reversed tactile and thermal hypersensitivity following axotomy, ligation or crush injury. Thermal and tactile hypersensitivity re-emerged within 1 week of treatment termination. However, artemin treated rats with nerve crush, but not axotomy or ligation, subsequently showed gradual return of sensory thresholds to pre-injury baseline levels by 6 weeks post-injury. Artemin normalized labeling for NF200, IB4 and CGRP in nerve fibers distal to the crush injury, suggesting persistent normalization of nerve-crush induced neurochemical changes. Sciatic and intradermal administration of dextran or CTB distal to the crush injury site resulted in labeling of neuronal profiles in the L5 DRG, suggesting functional restoration of non-myelinated and myelinated fibers across the injury site into cutaneous tissue. Artemin also diminished ATF3 and caspase-3 expression in the L5 DRG, suggesting persistent neuroprotective actions. A limited period of artemin treatment elicits disease modification by promoting sensory reinnervation of distal territories and restoring pre-injury sensory thresholds.
doi:10.1016/j.pain.2013.11.007
PMCID: PMC3936608  PMID: 24269493
Artemin; peripheral nerve injury; sensory neurons; hyperalgesia; regeneration
12.  Use of hybrid chitosan membranes and human mesenchymal stem cells from the Wharton jelly of umbilical cord for promoting nerve regeneration in an axonotmesis rat model★ 
Neural Regeneration Research  2012;7(29):2247-2258.
Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to assess the effect on nerve regeneration, associating a hybrid chitosan membrane with non-differentiated human mesenchymal stem cells isolated from Wharton's jelly of umbilical cord, in peripheral nerve reconstruction after crush injury. Chromosome analysis on human mesenchymal stem cell line from Wharton's jelly was carried out and no structural alterations were found in metaphase. Chitosan membranes were previously tested in vitro, to assess their ability in supporting human mesenchymal stem cell survival, expansion, and differentiation. For the in vivo testing, Sasco Sprague adult rats were divided in 4 groups of 6 or 7 animals each: Group 1, sciatic axonotmesis injury without any other intervention (Group 1-Crush); Group 2, the axonotmesis lesion of 3 mm was infiltrated with a suspension of 1 250–1 500 human mesenchymal stem cells (total volume of 50 μL) (Group 2-CrushCell); Group 3, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane covered with a monolayer of non-differentiated human mesenchymal stem cells (Group 3-CrushChitIIICell) and Group 4, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane (Group 4-CrushChitIII). Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index, static sciatic index, extensor postural thrust, and withdrawal reflex latency. Stereological analysis was carried out on regenerated nerve fibers. Results showed that infiltration of human mesenchymal stem cells, or the combination of chitosan membrane enwrapment and human mesenchymal stem cell enrichment after nerve crush injury provide a slight advantage to post-traumatic nerve regeneration. Results obtained with chitosan type III membrane alone confirmed that they significantly improve post-traumatic axonal regrowth and may represent a very promising clinical tool in peripheral nerve reconstructive surgery. Yet, umbilical cord human mesenchymal stem cells, that can be expanded in culture and induced to form several different types of cells, may prove, in future experiments, to be a new source of cells for cell therapy, including targets such as peripheral nerve and muscle.
doi:10.3969/j.issn.1673-5374.2012.29.002
PMCID: PMC4268725  PMID: 25538746
stem cells; mesenchymal stem cells; Wharton jelly; umbilical cord; biomaterials; chitosan; axonotmesis; functional analysis; rat; karyotype analysis; stereological analysis
13.  Accelerating axonal growth promotes motor recovery after peripheral nerve injury in mice 
The Journal of Clinical Investigation  2011;121(11):4332-4347.
Although peripheral nerves can regenerate after injury, proximal nerve injury in humans results in minimal restoration of motor function. One possible explanation for this is that injury-induced axonal growth is too slow. Heat shock protein 27 (Hsp27) is a regeneration-associated protein that accelerates axonal growth in vitro. Here, we have shown that it can also do this in mice after peripheral nerve injury. While rapid motor and sensory recovery occurred in mice after a sciatic nerve crush injury, there was little return of motor function after sciatic nerve transection, because of the delay in motor axons reaching their target. This was not due to a failure of axonal growth, because injured motor axons eventually fully re-extended into muscles and sensory function returned; rather, it resulted from a lack of motor end plate reinnervation. Tg mice expressing high levels of Hsp27 demonstrated enhanced restoration of motor function after nerve transection/resuture by enabling motor synapse reinnervation, but only within 5 weeks of injury. In humans with peripheral nerve injuries, shorter wait times to decompression surgery led to improved functional recovery, and, while a return of sensation occurred in all patients, motor recovery was limited. Thus, absence of motor recovery after nerve damage may result from a failure of synapse reformation after prolonged denervation rather than a failure of axonal growth.
doi:10.1172/JCI58675
PMCID: PMC3223863  PMID: 21965333
14.  Beneficial effects of treadmill training in experimental diabetic nerve regeneration 
Clinics  2010;65(12):1329-1337.
OBJECTIVES:
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.
MATERIALS AND METHOD:
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.
RESULTS:
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.
CONCLUSION:
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.
doi:10.1590/S1807-59322010001200017
PMCID: PMC3020345  PMID: 21340223
Diabetes; Sciatic nerve crush; Motor function; Nerve morphometry; Treadmill training
15.  Antiallodynic Effects of Acupuncture in Neuropathic Rats 
Yonsei Medical Journal  2006;47(3):359-366.
Peripheral nerve injury often results in abnormal neuropathic pain such as allodynia or hyperalgesia. Acupuncture, a traditional Oriental medicine, has been used to relieve pain and related symptoms. However, the efficiency of acupuncture in relieving neuropathic pain is not clear. The aim of this study was to investigate the anti-allodynic effects of acupuncture through behavioral and electrophysiological examinations. Male Sprague-Dawley rats were subjected to neuropathic surgery consisting of a tight ligation and transection of the left tibial and sural nerves, under pentobarbital anesthesia. The acupuncture experiment consisted of four different groups, one treated at each of three different acupoints (Zusanli (ST36), Yinlingquan (SP9), and a sham-acupoint) and a control group. Behavioral tests for mechanical allodynia and cold allodynia were performed for up to two weeks postoperatively. Extracellular electrophysiological recordings were made from the dorsal roots using platinum wire electrodes. Mechanical and cold allodynia were significantly reduced after acupuncture treatment at the Zusanli and Yinlingquan acupoints, respectively. Electrophysiological neural responses to von Frey and acetone tests were also reduced after acupuncture at the same two acupoints. These results suggest that acupuncture may be beneficial in relieving neuropathic pain.
doi:10.3349/ymj.2006.47.3.359
PMCID: PMC2688155  PMID: 16807985
Neuropathic pain; acupuncture; acupoint; allodynia; electrophysiology
16.  Improvement in acupoint selection for acupuncture of nerves surrounding the injury site: electro-acupuncture with Governor vessel with local meridian acupoints 
Neural Regeneration Research  2015;10(1):128-135.
Peripheral nerve injury not only affects the site of the injury, but can also induce neuronal apoptosis at the spinal cord. However, many acupuncture clinicians still focus only on the injury site, selecting acupoints entirely along the injured nerve trunk and neglecting other regions; this may delay onset of treatment efficacy and rehabilitation. Therefore, in the present study, we compared the clinical efficacy of acupuncture at Governor vessel and local meridian acupoints combined (GV/LM group) with acupuncture at local meridian acupoints alone (LM group) in the treatment of patients with peripheral nerve injury. In the GV/LM group (n = 15), in addition to meridian acupoints at the injury site, the following acupoints on the Governor vessel were stimulated: Baihui (GV20), Fengfu (GV16), Dazhui (GV14), and Shenzhu (GV12), selected to treat nerve injury of the upper limb, and Jizhong (GV6), Mingmen (GV4), Yaoyangguan (GV3), and Yaoshu (GV2) to treat nerve injury of the lower limb. In the LM group (n = 15), only meridian acupoints along the injured nerve were selected. Both groups had electroacupuncture treatment for 30 minutes, once a day, 5 times per week, for 6 weeks. Two cases dropped out of the LM group. A good or excellent clinical response was obtained in 80% of the patients in the GV/LM group and 38.5% of the LM group. In a second study, an additional 20 patients underwent acupuncture with the same prescription as the GV/LM group. Electomyographic nerve conduction tests were performed before and after acupuncture to explore the mechanism of action of the treatment. An effective response was observed in 80.0% of the patients, with greater motor nerve conduction velocity and amplitude after treatment, indicating that electroacupuncture on specific Governor vessel acupoints promotes functional motor nerve repair after peripheral nerve injury. In addition, electromyography was performed before, during and after electroacupuncture in one patient with radial nerve injury. After a single session, the patient's motor nerve conduction velocity increased by 23.2%, indicating that electroacupuncture at Governor vessel acupoints has an immediate therapeutic effect on peripheral nerve injury. Our results indicate that Governor vessel and local meridian acupoints used simultaneously promote functional repair after peripheral nerve injury. The mechanism of action may arise from an improvement of the local microenvironment in injured nervous tissue, as well as immediate effects of Governor vessel and local meridian acupoint stimulation to ensure the continuity between the peripheral and central nervous systems.
doi:10.4103/1673-5374.150720
PMCID: PMC4357096  PMID: 25788933
nerve regeneration; peripheral nerve injury; acupuncture; electroacupuncture; Governor vessel acupoints; local acupoints; electrophysiology; spinal cord; motor nerve conduction; functional repair; neural regeneration
17.  Regeneration of diabetic axons is enhanced by selective knockdown of the PTEN gene 
Brain  2014;137(4):1051-1067.
Neuropathy and impaired axonal regeneration are common complications of diabetes mellitus. Singh et al. reveal upregulation of the tumour suppressor PTEN in peripheral neurons in a mouse model of diabetes. Knockdown of PTEN by siRNA in vitro and in vivo enhanced the regeneration of diabetic axons following injury.
Diabetes mellitus renders both widespread and localized irreversible damage to peripheral axons while imposing critical limitations on their ability to regenerate. A major failure of regenerative capacity thereby imposes a ‘double hit’ in diabetic patients who frequently develop focal neuropathies such as carpal tunnel syndrome in addition to generalized diffuse polyneuropathy. The mechanisms of diabetic neuron regenerative failure have been speculative and few approaches have offered therapeutic opportunities. In this work we identify an unexpected but major role for PTEN upregulation in diabetic peripheral neurons in attenuating axon regrowth. In chronic diabetic neuropathy models in mice, we identified significant PTEN upregulation in peripheral sensory neurons of messenger RNA and protein compared to littermate controls. In vitro, sensory neurons from these mice responded to PTEN knockdown with substantial rises in neurite outgrowth and branching. To test regenerative plasticity in a chronic diabetic model with established neuropathy, we superimposed an additional focal sciatic nerve crush injury and assessed morphological, electrophysiological and behavioural recovery. Knockdown of PTEN in dorsal root ganglia ipsilateral to the side of injury was achieved using a unique form of non-viral short interfering RNA delivery to the ipsilateral nerve injury site and paw. In comparison with scrambled sequence control short interfering RNA, PTEN short interfering RNA improved several facets of regeneration: recovery of compound muscle action potentials, reflecting numbers of reconnected motor axons to endplates, conduction velocities of both motor and sensory axons, reflecting their maturation during regrowth, numbers and calibre of regenerating myelinated axons distal to the injury site, reinnervation of the skin by unmyelinated epidermal axons and recovery of mechanical sensation. Collectively, these findings identify a novel therapeutic approach, potentially applicable to other neurological conditions requiring specific forms of molecular knockdown, and also identify a unique target, PTEN, to treat diabetic neuroregenerative failure.
doi:10.1093/brain/awu031
PMCID: PMC3959560  PMID: 24578546
diabetic polyneuropathy; diabetes; peripheral nervous system; peripheral nerve; axonal injury
18.  Brain-Derived Neurotrophic Factor from Bone Marrow-Derived Cells Promotes Post-Injury Repair of Peripheral Nerve 
PLoS ONE  2012;7(9):e44592.
Brain-derived neurotrophic factor (BDNF) stimulates peripheral nerve regeneration. However, the origin of BNDF and its precise effect on nerve repair have not been clarified. In this study, we examined the role of BDNF from bone marrow-derived cells (BMDCs) in post-injury nerve repair. Control and heterozygote BDNF knockout mice (BDNF+/−) received a left sciatic nerve crush using a cerebral blood clip. Especially, for the evaluation of BDNF from BMDCs, studies with bone marrow transplantation (BMT) were performed before the injury. We evaluated nerve function using a rotarod test, sciatic function index (SFI), and motor nerve conduction velocity (MNCV) simultaneously with histological nerve analyses by immunohistochemistry before and after the nerve injury until 8 weeks. BDNF production was examined by immunohistochemistry and mRNA analyses. After the nerve crush, the controls showed severe nerve dysfunction evaluated at 1 week. However, nerve function was gradually restored and reached normal levels by 8 weeks. By immunohistochemistry, BDNF expression was very faint before injury, but was dramatically increased after injury at 1 week in the distal segment from the crush site. BDNF expression was mainly co-localized with CD45 in BMDCs, which was further confirmed by the appearance of GFP-positive cells in the BMT study. Variant analysis of BDNF mRNA also confirmed this finding. BDNF+/− mice showed a loss of function with delayed histological recovery and BDNF+/+→BDNF+/− BMT mice showed complete recovery both functionally and histologically. These results suggested that the attenuated recovery of the BDNF+/− mice was rescued by the transplantation of BMCs and that BDNF from BMDCs has an essential role in nerve repair.
doi:10.1371/journal.pone.0044592
PMCID: PMC3446933  PMID: 23028564
19.  Retrograde tracing and toe spreading after experimental autologous nerve transplantation and crush injury of the sciatic nerve: a descriptive methodological study 
Evaluation of functional and structural recovery after peripheral nerve injury is crucial to determine the therapeutic effect of a nerve repair strategy. In the present study, we examined the relationship between the structural evaluation of regeneration by means of retrograde tracing and the functional analysis of toe spreading. Two standardized rat sciatic nerve injury models were used to address this relationship. As such, animals received either a 2 cm sciatic nerve defect (neurotmesis) followed by autologous nerve transplantation (ANT animals) or a crush injury with spontaneous recovery (axonotmesis; CI animals). Functional recovery of toe spreading was observed over an observation period of 84 days. In contrast to CI animals, ANT animals did not reach pre-surgical levels of toe spreading. After the observation period, the lipophilic dye DiI was applied to label sensory and motor neurons in dorsal root ganglia (DRG; sensory neurons) and spinal cord (motor neurons), respectively. No statistical difference in motor or sensory neuron counts could be detected between ANT and CI animals.
In the present study we could indicate that there was no direct relationship between functional recovery (toe spreading) measured by SSI and the number of labelled (motor and sensory) neurons evaluated by retrograde tracing. The present findings demonstrate that a multimodal approach with a variety of independent evaluation tools is essential to understand and estimate the therapeutic benefit of a nerve repair strategy.
doi:10.1186/1749-7221-7-5
PMCID: PMC3473253  PMID: 22546145
Peripheral nerve injury; Repair strategy; Peripheral nerve regeneration; Neurotmesis; SSI; Sciatic nerve injury; Rat model
20.  Acupuncture and vitamin B12 injection for Bell’s palsy: no high-quality evidence exists 
Neural Regeneration Research  2015;10(5):808-813.
OBJECTIVE:
To assess the efficacy of acupuncture combined with vitamin B12 acupoint injection versus acupuncture alone to reduce incomplete recovery in patients with Bell’s palsy.
DATA RETRIEVAL:
A computer-based online retrieval of Medline, Web of Science, CNKI, CBM databases until April 2014 was performed for relevant trials, using the key words “Bell’s palsy or idiopathic facial palsy or facial palsy” and “acupuncture or vitamin B12 or methylcobalamin”.
STUDY SELECTION:
All randomized controlled trials that compared acupuncture with acupuncture combined with vitamin B12 in patients with Bell’s palsy were included in the meta-analysis. The initial treatment lasted for at least 4 weeks. The outcomes of incomplete facial recovery were monitored. The scoring index varied and the definition of healing was consistent. The combined effect size was calculated by using relative risk (RR) with 95% confidence interval (CI) using the fixed effect model of Review Manager.
MAIN OUTCOME MEASURES:
Incomplete recovery rates were chosen as the primary outcome.
RESULTS:
Five studies involving 344 patients were included in the final analysis. Results showed that the incomplete recovery rate of Bell’s palsy patients was 44.50% in the acupuncture combined with vitamin B12 group but 62.57% in the acupuncture alone group. The major acupoints were Taiyang (EX-HN5), Jiache (ST6), Dicang (ST4) and Sibai (ST2). The combined effect size showed that acupuncture combined with vitamin B12 was better than acupuncture alone for the treatment of Bell’s palsy (RR = 0.71, 95%CI: 0.58–0.87; P = 0.001), this result held true when 8 patients lost to follow up in one study were included into the analyses (RR = 0.70, 95%CI: 0.58–0.86; P = 0.0005). In the subgroup analyses, the therapeutic effect in patients of the electroacupuncture subgroup was better than in the non-electroacupuncture subgroup (P = 0.024). There was no significant difference in the incomplete recovery rate by subgroup analysis on drug types and treatment period. Most of the included studies were moderate or low quality, and bias existed.
CONCLUSION:
In patients with Bell’s palsy, acupuncture combined with vitamin B12 can reduce the risk of incomplete recovery compared with acupuncture alone in our meta-analysis. Because of study bias and methodological limitations, this conclusion is uncertain and the clinical application of acupuncture combined with vitamin B12 requires further exploration.
doi:10.4103/1673-5374.156987
PMCID: PMC4468776  PMID: 26109959
nerve regeneration; brain injury; facial palsy; Bell's palsy; comparison; methodological quality; therapy; fixed effect model; acupuncture; incomplete recovery; randomized controlled trials; electroacupuncture; NSFC grants; neural regeneration
21.  Electro-acupuncture for fatigue, sleep, and psychological distress in breast cancer patients with aromatase inhibitor-related arthralgia: A randomized trial 
Cancer  2014;120(23):3744-3751.
Purpose
Although fatigue, sleep disturbance, depression, and anxiety are associated with pain in breast cancer patients, it is unknown if acupuncture can decrease these co-morbid symptoms in cancer patients with pain. This study aimed at evaluating the effect of electro-acupuncture on fatigue, sleep, and psychological distress in breast cancer survivors who experience joint pain related to aromatase inhibitors (AIs).
Patients and methods
We performed a randomized controlled trial of an eight-week course of electro-acupuncture (EA) as compared to waitlist control (WLC) and sham acupuncture (SA) in postmenopausal women with breast cancer who self-reported joint pain attributable to aromatase inhibitors. Fatigue, sleep disturbance, anxiety, and depression were measured by the Brief Fatigue Inventory (BFI), Pittsburgh Sleep Quality Index (PSQI), and Hospital Anxiety and Depression Scale (HADS). The effects of EA and SA vs. WLC on these outcomes were evaluated using mixed-effects models.
Results
Of the 67 randomly assigned patients, baseline pain interference was associated with fatigue (Pearson correlation coefficient r =0.75, p<0.001), sleep disturbance (r=0.38, p=0.0026), and depression (r= 0.58, p<0.001). Compared to the WLC, EA produced significant improvement in fatigue (p=0.0095), anxiety (p=0.044), and depression (p=0.015) and non-significant improvement in sleep disturbance (p=0.058) during the 12 week intervention and follow up period. In contrast, SA did not produce significant reduction in fatigue and anxiety symptoms, but produced significant improvement in depression compared with WLC (p=0.0088).
Conclusion
Compared to usual care, EA produced significant improvement in fatigue, anxiety, and depression, whereas SA improved only depression in women experiencing AI-related arthralgia.
Clinical Trial Registration
NCT01013337
doi:10.1002/cncr.28917
PMCID: PMC4239308  PMID: 25077452
Acupuncture; breast neoplasm; Aromatase inhibitors; fatigue; sleep; psychological distress
22.  Enhanced Immune Response in Immunodeficient Mice Improves Peripheral Nerve Regeneration Following Axotomy 
Injuries to peripheral nerves cause loss of motor and sensory function, greatly affecting life quality. Successful repair of the lesioned nerve requires efficient cell debris removal, followed by axon regeneration and reinnervation of target organs. Such process is orchestrated by several cellular and molecular events in which glial and immune cells actively participate. It is known that tissue clearance is largely improved by macrophages, which activation is potentiated by cells and molecules of the acquired immune system, such as T helper lymphocytes and antibodies, respectively. In the present work, we evaluated the contribution of lymphocytes in the regenerative process of crushed sciatic nerves of immunocompetent (wild-type, WT) and T and B-deficient (RAG-KO) mice. In Knockout animals, we found increased amount of macrophages under basal conditions and during the initial phase of the regenerative process, that was evaluated at 2, 4, and 8 weeks after lesion (wal). That parallels with faster axonal regeneration evidenced by the quantification of neurofilament and a growth associated protein immunolabeling. The motor function, evaluated by the sciatic function index, was fully recovered in both mouse strains within 4 wal, either in a progressive fashion, as observed for RAG-KO mice, or presenting a subtle regression, as seen in WT mice between 2 and 3 wal. Interestingly, boosting the immune response by early adoptive transference of activated WT lymphocytes at 3 days after lesion improved motor recovery in WT and RAG-KO mice, which was not ameliorated when cells were transferred at 2 wal. When monitoring lymphocytes by in vivo imaging, in both mouse strains, cells migrated to the lesion site shortly after transference, remaining in the injured limb up to its complete motor recovery. Moreover, a first peak of hyperalgesia, determined by von-Frey test, was coincident with increased lymphocyte infiltration in the damaged paw. Overall, the present results suggest that a wave of immune cell infiltration takes place during subacute phase of axonal regeneration, resulting in transient set back of motor recovery following peripheral axonal injury. Moreover, modulation of the immune response can be an efficient approach to speed up nerve regeneration.
doi:10.3389/fncel.2016.00151
PMCID: PMC4905955  PMID: 27378849
sciatic nerve; PNS; neuroimmunology; inflammation; axonal regeneration
23.  Misdirection of regenerating motor axons after nerve injury and repair in the rat sciatic nerve model 
Experimental neurology  2008;211(2):339-350.
Misdirection of regenerating axons is one of the factors that can explain the poor results often found after nerve injury and repair. In this study, we quantified the degree of misdirection and the effect on recovery of function after different types of nerve injury and repair in the rat sciatic nerve model; crush injury, direct coaptation, and autograft repair. Sequential tracing with retrograde labeling of the peroneal nerve before and 8 weeks after nerve injury and repair was performed to quantify the accuracy of motor axon regeneration. Digital video analysis of ankle motion was used to investigate the recovery of function. In addition, serial compound action potential recordings and nerve and muscle morphometry were performed. In our study, accuracy of motor axon regeneration was found to be limited; only 71% (±4.9%) of the peroneal motoneurons were correctly directed 2 months after sciatic crush injury, 42% (±4.2%) after direct coaptation, and 25% (±6.6%) after autograft repair. Recovery of ankle motion was incomplete after all types of nerve injury and repair and demonstrated a disturbed balance of ankle plantar and dorsiflexion. The number of motoneurons from which axons had regenerated was not significantly different from normal. The number of myelinated axons was significantly increased distal to the site of injury. Misdirection of regenerating motor axons is a major factor in the poor recovery of nerves that innervate different muscles. The results of this study can be used as basis for developing new nerve repair techniques that may improve the accuracy of regeneration.
doi:10.1016/j.expneurol.2007.12.023
PMCID: PMC2967197  PMID: 18448099
Aberrant reinnervation; Accuracy of regeneration; Ankle motion analysis; Double labeling; Sequential retrograde tracing
24.  A Combination of Schwann-Cell Grafts and Aerobic Exercise Enhances Sciatic Nerve Regeneration 
PLoS ONE  2014;9(10):e110090.
Background
Despite the regenerative potential of the peripheral nervous system, severe nerve lesions lead to loss of target-organ innervation, making complete functional recovery a challenge. Few studies have given attention to combining different approaches in order to accelerate the regenerative process.
Objective
Test the effectiveness of combining Schwann-cells transplantation into a biodegradable conduit, with treadmill training as a therapeutic strategy to improve the outcome of repair after mouse nerve injury.
Methods
Sciatic nerve transection was performed in adult C57BL/6 mice; the proximal and distal stumps of the nerve were sutured into the conduit. Four groups were analyzed: acellular grafts (DMEM group), Schwann cell grafts (3×105/2 µL; SC group), treadmill training (TMT group), and treadmill training and Schwann cell grafts (TMT + SC group). Locomotor function was assessed weekly by Sciatic Function Index and Global Mobility Test. Animals were anesthetized after eight weeks and dissected for morphological analysis.
Results
Combined therapies improved nerve regeneration, and increased the number of myelinated fibers and myelin area compared to the DMEM group. Motor recovery was accelerated in the TMT + SC group, which showed significantly better values in sciatic function index and in global mobility test than in the other groups. The TMT + SC group showed increased levels of trophic-factor expression compared to DMEM, contributing to the better functional outcome observed in the former group. The number of neurons in L4 segments was significantly higher in the SC and TMT + SC groups when compared to DMEM group. Counts of dorsal root ganglion sensory neurons revealed that TMT group had a significant increased number of neurons compared to DMEM group, while the SC and TMT + SC groups had a slight but not significant increase in the total number of motor neurons.
Conclusion
These data provide evidence that this combination of therapeutic strategies can significantly improve functional and morphological recovery after sciatic injury.
doi:10.1371/journal.pone.0110090
PMCID: PMC4198198  PMID: 25333892
25.  Thermographic evaluation of hind paw skin temperature and functional recovery of locomotion after sciatic nerve crush in rats 
Clinics  2011;66(7):1259-1266.
INTRODUCTION:
Peripheral nerves are often damaged by direct mechanical injury, diseases, and tumors. The peripheral nerve injuries that result from these conditions can lead to a partial or complete loss of motor, sensory, and autonomic functions, which in turn are related to changes in skin temperature, in the involved segments of the body. The aim of this study was to evaluate the changes in hind paw skin temperature after sciatic nerve crush in rats in an attempt to determine whether changes in skin temperature correlate with the functional recovery of locomotion.
METHODS:
Wistar rats were divided into three groups: control (n = 7), sham (n = 25), and crush (n = 25). All groups were subjected to thermographic, functional, and histological assessments.
RESULTS:
ΔT in the crush group was different from the control and sham groups at the 1st, 3rd and 7rd postoperative days (p<0.05). The functional recovery from the crush group returned to normal values between the 3rd and 4th week post-injury, and morphological analysis of the nerve revealed incomplete regeneration at the 4th week after injury.
DISCUSSION:
This study is the first demonstration that sciatic nerve crush in rats induces an increase in hind paw skin temperature and that skin temperature changes do not correlate closely with functional recovery
doi:10.1590/S1807-59322011000700023
PMCID: PMC3148474  PMID: 21876984
Thermography; Skin temperature; Nerve crush; Functional recovery

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