Effect of low-intensity millimeter wave electromagnetic radiation on regeneration of the sciatic nerve in rats

The effect of low-intensity millimeter wave electromagnetic radiation (MWR) on regeneration of the rat sciatic nerve after transection and microsurgical reapproximation was examined. Rats were exposed to 54 GHz MWR at a power density of 4 mW/cm². It was found that MWR treatment of the femoral skin in the area of suture accelerated the regeneration of nerve fibers. At the twentieth postoperative day, the MWR-treated animals had a 32% increase in the regeneration distance compared to the control animals. The conduction velocity showed a 26% increase in the MWR-treated animals. © 1996 Wiley-Liss, Inc.     

Pulsed electromagnetic fields induce peripheral nerve regeneration and endplate enzymatic changes

An experimental study was carried out in rats with the purpose of demonstrating the capacity of pulsed electromagnetic fields (PEMFs) to stimulate regeneration of the peripheral nervous system (PNS). Wistar and Brown Norway (BN) rats were used. Direct sciatic nerve anastomoses were performed after section or allograft interposition. Treatment groups then received 4 weeks of PEMFs. Control groups received no stimulation. The evaluation of the results was carried out by quantitative morphometric analysis, demonstrating a statistically significant increase in regeneration indices (P < 0.05) in the stimulated groups (9000 +/- 5000 and 4000 +/- 6000) compared to the non-stimulated groups (2000 +/- 4000 and 700 +/- 200). An increase of NAD specific isocitrate dehydrogenase (IDH) activity was found along with an increase in the activity of acetyl cholinesterase at the motor plate. The present study might lead to the search for new alternatives in the stimulation of axonal regenerative processes in the PNS and other possible clinical applications.     

大鼠坐骨神经挤压损伤导致脊髓单突触反射回返性抑制的长时程减弱

坐骨神经损伤是临床常见的周围神经疾病。神经损伤后再生肌肉和运动神经元会出现各种功能障碍,虽然其中一部分因素已被阐明,但多局限于受损神经局部,而对于再生后脊髓运动神经元的回返性抑制(recurrent inhibition,RI)通路的功能变化却很少被报道。本文研究大鼠短暂坐骨神经损伤后,恢复神经再支配(reinnervation)情况下,脊髓RI通路的功能变化。在正常或坐骨神经挤压(crush)受损后的成年大鼠上,通过刺激离断的脊髓背根(L5),在外侧腓肠肌-比目鱼肌(lateral gas-trocnemius-soleus,LG-S)神经或内侧腓肠肌(medial gastrocnemius,MG)神经记录单突触反射(monosynaptic reflex,MSR),并同时在另一神经给予条件性刺激,以检测LG-S和MG运动神经元间RI的变化。结果显示:(1)脊髓运动神经元的RI在坐骨神经挤压受损后即基本丢失(<5周),至损伤6周后部分恢复至正常的50%,并至少维持至损伤14周后;(2)一侧的坐骨神经损伤对对侧的RI没有影响;(3)外周神经损伤后,免疫组织化学方法显示脊髓运动神经元数目本身并不发生减少。以上...     

Extracellular vesicles from human umbilical cord mesenchymal stem cells improve nerve regeneration after sciatic nerve transection in rats

Peripheral nerve injury results in limited nerve regeneration and severe functional impairment. Mesenchymal stem cells (MSCs) are a remarkable tool for peripheral nerve regeneration. The involvement of human umbilical cord MSC‐derived extracellular vesicles (hUCMSC‐EVs) in peripheral nerve regeneration, however, remains unknown. In this study, we evaluated functional recovery and nerve regeneration in rats that received hUCMSC‐EV treatment after nerve transection. We observed that hUCMSC‐EV treatment promoted the recovery of motor function and the regeneration of axons; increased the sciatic functional index; resulted in the generation of numerous axons and of several Schwann cells that surrounded individual axons; and attenuated the atrophy of the gastrocnemius muscle. hUCMSC‐EVs aggregated to rat nerve defects, down‐regulated interleukin (IL)‐6 and IL‐1β, up‐regulated IL‐10 and modulated inflammation in the injured nerve. These effects likely contributed to the promotion of nerve regeneration. Our findings indicate that hUCMSC‐EVs can improve functional recovery and nerve regeneration by providing a favourable microenvironment for nerve regeneration. Thus, hUCMSC‐EVs have considerable potential for application in the treatment of peripheral nerve injury.     

Acceleration of axonal outgrowth in rat sciatic nerve at one week after axotomy

Following injury of sciatic motor axons in the rat, the rate of axonal outgrowth is faster if there has been a prior “conditioning” axotomy. The acceleration of outgrowth is due to an acceleration of SCb, the rate [slow (SC)] component of axonal transport that carries cytomatrix proteins; this occurs throughout the axon by 7 days after the conditioning axotomy (Jacob and McQuarrie, 1991a, J. Neurobiol. 22:570–583). To further characterize the conditioning lesion effect (CLE), it is important to know (1) the minimum effective conditioning interval (time between conditioning and testing lesions), (2) whether the cell body reaction is required, and (3) whether outgrowth accelerates after a single axotomy. Outgrowth distances were measured by radiolabeling all newly synthesized neuronal proteins and detecting those carried to growth cones by fast axonal transport. When the conditioning and testing lesions were made simultaneously (0 day conditioning interval), there was no CLE. With a conditioning interval of 3 days, there was a shortening of the initial delay (before the onset of outgrowth) without a change in outgrowth rate. With conditioning intervals of 7, 14, and 21 days, the rates of outgrowth were increased by 8%, 22%, and 11%, respectively. To determine whether the cell body reaction to axotomy is necessary for the CLE, a nonaxotomizing stimulus to axonal growth (partial denervation) was used in place of a conditioning axotomy. This had no effect on the rate of outgrowth from a testing lesion made 14 days later. Finally, we examined the possibility that outgrowth accelerates after a single lesion. Outgrowth was faster at 6–9 days after axotomy than at 3–6 days (p < 0.001), and accelerated further at 9–12 days (p < 0.001). We conclude that (1) the shortest effective conditioning interval is 3 days; (2) the cell body reaction is necessary for the CLE; (3) axonal outgrowth from a single axotomy accelerates in concert with the anabolic phase of the cell body reaction. The SCb motor is, in turn, upregulated by this reaction. This suggests that the SCb motor responds to a fast-transported signal that is a product of the cell body reaction. © 1993 John Wiley & Sons, Inc.     

GDNF-ADSCs-APG embedding enhances sciatic nerve regeneration after electrical injury in a rat model

The pluripotency of adipose-derived stem cells (ADSCs) makes them appropriate for tissue repair and wound healing. Owing to the repair properties of autologous platelet–rich gel (APG), which is based on easily accessible blood platelets, its clinical use has been increasingly recognized by physicians. The aim of this study was to investigate the effect of combined treatment with ADSCs and APG on sciatic nerve regeneration after electrical injury. To facilitate the differentiation of ADSCs, glial cell line–derived neurotrophic factor (GDNF) was overexpressed in ADSCs by lentivirus transfection. GDNF-ADSCs were mingled with APG gradient concentrations, and in vitro, cell proliferation and differentiation were examined with 5-ethynyl-2′-deoxyuridine staining and immunofluorescence. A rat model was established by exposing the sciatic nerve to an electrical current of 220 V for 3 seconds. Rat hind-limb motor function and sciatic nerve regeneration were subsequently evaluated. Rat ADSCs were characterized by high expression of CD90 and CD105, with scant expression of CD34 and CD45. We found that GDNF protein expression in ADSCs was elevated after Lenti-GDNF transfection. In GDNF-ADSCs-APG cultures, GDNF was increasingly produced while tissue growth factor-β was reduced as incubation time was increased. ADSC proliferation was augmented and neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) expression were upregulated in GDNF-ADSCs-APG. In addition, limb motor function and nerve axon growth were improved after GDNF-ADSCs-APG treatment. In conclusion, our study demonstrates the combined effect of ADSCs and APG in peripheral nerve regeneration and may lead to treatments that benefit patients with electrical injuries.     

Effect of weak, interrupted sinusoidal low frequency magnetic field on neural regeneration in rats: functional evaluation

A study of the effect of weak, interrupted sinusoidal low frequency magnetic field (ISMF) stimulation on regeneration of the rat sciatic nerve was carried out. In the experiment, 60 Wistar rats were used: 24 rats underwent unilateral sciatic nerve transection injury and immediate surgical nerve repair, 24 rats underwent unilateral sciatic nerve crush injury, and the remaining 12 rats underwent a sham surgery. Half of the animals (n = 12) with either sciatic nerve lesion were randomly chosen and exposed between a pair of Helmholtz coils for 3 weeks post-injury, 4 h/day, to an interrupted (active period to pause ratio = 1.4 s/0.8 s) sinusoidal 50 Hz magnetic field of 0.5 mT. The other half of the animals (n = 12) and six rats with sham surgery were used for two separate controls. Functional recovery was followed for 6 weeks for the crush injuries and 7(1/2) months for the transection injuries by video assisted footprint analysis in static conditions and quantified using a recently revised static sciatic index (SSI) formula. We ascertained that the magnetic field influence was weak, but certainly detectable in both injury models. The accuracy of ISMF influence detection, determined by the one-way repeated measures ANOVA test, was better for the crush injury model: F(1, 198) = 9.0144, P = .003, than for the transection injury model: F(1, 198) = 6.4826, P = .012. The Student-Newman-Keuls range test for each response day yielded significant differences (P < .05) between the exposed and control groups early in the beginning of functional recovery and later on from the points adjacent to the beginning of the plateau, or 95% of functional recovery, and the end of observation. These differences probably reflect the ISMF systemic effect on the neuron cell bodies and increased and more efficient reinnervation of the periphery.     

Variable spatial magnetic field influences peripheral nerves regeneration in rats

Generator of spatial magnetic field is one of most recent achievements among the magnetostimulators. This apparatus allows to obtain the rotating magnetic field. This new method may be more effective than other widely used techniques of magnetostimulation and magnetotherapy. We investigated the influence of alternating, spatial magnetic field on the regeneration of the crushed rat sciatic nerves. Functional and morphological evaluations were used. After crush injury of the right sciatic nerve, Wistar C rats (n?=?80) were randomly divided into four groups (control and three experimental). The experimental groups (A, B, C) were exposed (20?min/day, 5?d/week, 4 weeks) to alternating spatial magnetic field of three different intensities. Sciatic Functional Index (SFI) and tensometric assessments were performed every week after nerve crush. Forty-eight hours before the sacrificing of animals, DiI (1,1’-di-octadecyl-3,3,3’,3’-tetramethyloindocarbocyanine perchlorate) was applied 5?mm distally to the crush site. Collected nerves and dorsal root ganglia (DRG) were subjected to histological and immunohistochemical staining. The survival rate of DRG neurons was estimated. Regrowth and myelination of the nerves was examined. The results of SFI and tensometric assessment showed improvement in all experimental groups as compared to control, with best outcome observed in group C, exposed to the strongest magnetic field. In addition, DRG survival rate and nerve regeneration intensity were significantly higher in the C group. Above results indicate that strong spatial alternating magnetic field exerts positive effect on peripheral nerve regeneration and its application could be taken under consideration in the therapy of injured peripheral nerves.     

Non-thermal plasma application enhances the recovery of transected sciatic nerves in rats

This experimental research aimed to investigate the effects of non-thermal plasma on nerve regeneration after transected nerve damage using the sciatic nerve in Wistar albino (A) rats. The experiments were performed on 27 Wistar A rats. The rats underwent surgery for right sciatic nerve exposure and were divided into three groups (each group, n = 9) according to sciatic nerve transected injury (SNTI) and non-thermal plasma application: a non-nerve damage (non-ND) group, a only nerve damage without non-thermal plasma application (ND) group, and a nerve damage with non-thermal plasma application (ND + NTP) group. Subsequent to SNTI and immediate suture, non-thermal plasma was administered three times per week for eight weeks. Evaluation for functional recovery was performed using the static sciatic index measured over the full treatment period of eight weeks. The sciatic nerve specimens were obtained after euthanasia and third day from the last non-thermal plasma application. The sciatic nerve tissues were subjected to histological analysis. Behavior analysis presented that the ND + NTP group showed improved static sciatic index compared with the nerve damage group. Histopathological findings demonstrated that the ND + NTP group had more dense Schwann cells and well-established continuity of nerve fibers, greater than the nerve damage group. Immunohistochemistry showed that the ND + NTP group had increased levels of markers for microtubule-associated protein 2 (MAP2), tau, S100 calcium-binding protein B, and neurofilament-200 and regulated the overexpression of CD68 and MAP2. These results indicated that non-thermal plasma enhanced the motor function and restored the neuronal structure by accelerating myelination and axonal regeneration. Additionally, non-thermal plasma was confirmed to have a positive effect on the recovery of SNTI in rats.     

Prospects on clinical applications of electrical stimulation for nerve regeneration

Regenerative capability is limited in higher vertebrates but present in organ systems such as skin, liver, bone, and to some extent, the nervous system. Peripheral nerves in particular have a relatively high potential for regeneration following injury. However, delay in regrowth or growth, blockage, or misdirection at the injury site, and growth to inappropriate end organs may compromise successful regeneration, leading to poor clinical results. Recent studies indicate that low-intensity electrical stimulation is equivalent to various growth factors, offering avenues to improve these outcomes. We present a review of studies using electric and electromagnetic fields that provide evidence for the enhancement of regeneration following nerve injury. Electric and electromagnetic fields (EMFs) have been used to heal fracture non-unions. This technology emerged as a consequence of basic studies [Yasuda, 1953; Fukada and Yasuda, 1957] demonstrating the piezoelectric properties of (dry) bone. The principle for using electrical stimulation for bone healing originated from the work of Bassett and Becker [1962], who described asymmetric voltage waveforms from mechanically deformed live bone. These changes were presumed to occur in bone during normal physical activity as a result of mechanical forces, and it was postulated that these forces were linked to modifications in bone structure. Endogenous currents present in normal tissue and those that occur after injury were proposed to modify bone structure [Bassett, 1989]. These investigators proposed that tissue integrity and function could be restored by applying electrical and/or mechanical energy to the area of injury. They successfully applied electrical currents to nonhealing fractures (using surgically implanted electrodes or pulsed currents using surface electrodes) to aid endogenous currents in the healing process. A considerable technological improvement was made with the noninvasive application of EMFs [Bassett et al., 1974] to accelerate fracture repair. This newer technique allowed the treatment of hard tissues without the complications of invasive electrode insertion. In addition, soft tissue injuries were now accessible for treatment by electromagnetic fields. In this article, we will first define the basic problems encountered in nerve injury and regeneration, and then review both in vitro and in vivo studies on the use of electric and electromagnetic fields to stimulate the healing process.     

Transthyretin enhances nerve regeneration

Mutations in transthyretin (TTR) are associated with familial amyloid polyneuropathy, a neurodegenerative disorder characterized by TTR deposition in the PNS. The aim of this study was to unravel whether TTR has a role in nerve physiology that could account for its preferential accumulation in the PNS, when mutated. The sensorimotor performance of wild-type and TTR knockout (KO) littermate mice was compared and showed impairment in mice lacking TTR. Given the possibility that, upon regeneration, the consequences arising from TTR absence might be exacerbated, nerve crush was performed in both strains. TTR KO mice presented delayed functional recovery resulting from decreased number of myelinated and unmyelinated fibers. Moreover, in transgenic mice in a TTR KO background, expressing human TTR in neurons, this phenotype was rescued, reinforcing that TTR enhances nerve regeneration. In vitro assays showed that neurite outgrowth and extension were decreased in the absence of TTR, probably underlying the decreased number of regenerating axons in TTR KO mice. Our findings demonstrate that TTR participates in nerve physiology and that it enhances nerve regeneration. Moreover, the assignment of a TTR function in nerve biology and repair, may explain its preferential deposition, when mutated, in the PNS of familial amyloid polyneuropathy patients.     

Faster nerve regeneration after sciatic nerve injury in mice over‐expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF‐2) is expressed in the peripheral nervous system and is up‐regulated after nerve lesion. It has been demonstrated that administration of FGF‐2 protects neurons from injury‐induced cell death and promotes axonal regrowth. Using transgenic mice over‐expressing FGF‐2 (TgFGF‐2), we addressed the importance of endogenously generated FGF‐2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild‐type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF‐2. Morphometric evaluation of intact nerves from TgFGF‐2 mice revealed no difference in number and size of myelinated fibers compared to wild‐type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF‐2 over‐expression on Schwann cell proliferation during the early regeneration process, we used BrdU‐labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild‐types. We propose that endogenously synthesized FGF‐2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Ultracytochemical identification of catecholamine-containing vesicles in the ligated sciatic nerve of the rat. Comparison with sympathetic nerve terminals

Summary Using the fixation procedure of Tranzer, three kinds of granular vesicles were identified in certain unmyelinated fibres of rat sciatic nerves proximal to a ligature: (1) small vesicles (SGV: 30–60 nm in diameter), (2) large vesicles (LGV: 60–100nm in diameter), and (3) large elongated vesicles (LEV: 60–100nm in diameter). A comparative study concerning the distribution of these granular vesicles was carried out using a cytopharmacological method (reserpine) and employing different fixatives (aldehydes + OsO₄, or OsO₄ alone) in periarterial nerve plexus of the femoral artery, vas deferens and the pineal organ.Use of Tranzer's method allows preservation in almost all granular vesicles of a strongly electron-dense core, while with the other fixatives mainly small, eccentric dense cores occur in the vesicles. Two main features were observed in ligated sciatic nerves: (i) a clear increase in the number of LGV, and (ii) the presence of LEV, considered as a variety of LGV rather than a new population of granular vesicles. Reserpine caused the cores of SGV to disappear almost completely, while LGV and LEV remained only partly depleted. The original method combining Tranzer's fixation procedure with radioautography revealed radioautographic labelling only in the unmyelinated fibres of ligated sciatic nerves and mainly superimposed over SGV, LGV and LEV. It is suggested that (i) SGV, LGV and also LEV represent possible storage sites of catecholamines, and (ii) a local morphogenesis of SGV from the large vesicles occurs in ligated sympathetic nerve fibres.     

A nanofibrous PHBV tube with Schwann cell as artificial nerve graft contributing to Rat sciatic nerve regeneration across a 30-mm defect bridge

Abstract

A nanofibrous PHBV nerve conduit has been used to evaluate its efficiency based on the promotion of nerve regeneration in rats. The designed conduits were investigated by physical, mechanical and microscopic analyses. The conduits were implanted into a 30-mm gap in the sciatic nerves of the rats. Four months after surgery, the regenerated nerves were evaluated by macroscopic assessments and histology. This polymeric conduit had sufficiently high mechanical properties to serve as a nerve guide. The results demonstrated that in the nanofibrous graft with cells, the sciatic nerve trunk had been reconstructed with restoration of nerve continuity and formatted nerve fibers with myelination. For the grafts especially the nanofibrous conduits with cells, muscle cells of gastrocnemius on the operated side were uniform in their size and structures. This study proves the feasibility of artificial conduit with Schwann cells for nerve regeneration by bridging a longer defect in a rat model.     

Studies of exposure of rabbits to electromagnetic pulsed fields

Dutch rabbits were acutely exposed to electromagnetic pulsed (EMP) fields (pulse duration 0.4μs, field strengths of 1–2 kV/cm and pulse repetition rates in the range of 10 to 38 Hz) for periods of up to two hours. The dependent variables investigated were pentobarbital-induced sleeping time and serum chemistry (including serum triglycerides, creatine phosphokinase (CPK) isoenzymes, and sodium and potassium). Core temperature measured immediately pre-exposure and postexposure revealed no exposure-related alterations. Over the range of field strengths and pulse durations investigated no consistent, statistically significant alterations were found in the end-points investigated.     

NgR RNA interference, combined with zymosan intravitreal injection, enhances optic nerve regeneration

Mature retinal ganglion cells like other CNS neurons are unable to regenerate their axons after injury. Regenerative failure has been attributed, in part, to two factors: the existence of myelin-derived inhibitors that bind to the Nogo receptor (NgR) and a deficiency of trophic support factors. We investigated the regrowth of injured axons both by inhibiting NgR by RNA interference and by recruiting exogenous trophic support by zymosan intravitreal injection. Our results showed that either approach can stimulate optic nerve axon regrowth but regenerated axons can grow longer and extend further when both methods are combined. We conclude that endogenous NgR inhibition and exogenous trophic support both play independent, important roles in enhancing optic nerve axon regrowth and that the regenerative effect can be augmented when the two are combined. This may provide a therapeutic strategy for promoting axon regeneration in the CNS as well.     

Exosomes from human adipose-derived stem cells promote sciatic nerve regeneration via optimizing Schwann cell function

Human adipose-derived stem cells (ASCs) have a potential for the treatment of peripheral nerve injury. Recent studies demonstrated that stem cells can mediate therapeutic effect by secreting exosomes. We aimed to investigate the effect of human ASCs derived exosomes (ASC-Exos) on peripheral nerve regeneration in vitro and in vivo. Our results showed after being internalized by Schwann cells (SCs), ASC-Exos significantly promoted SC proliferation, migration, myelination, and secretion of neurotrophic factors by upregulating corresponding genes in vitro. We next evaluated the efficacy of ASC-Exo therapy in a rat sciatic nerve transection model with a 10-mm gap. Axon regeneration, myelination, and restoration of denervation muscle atrophy in ASC-Exos treated group was significantly improved compared to vehicle control. This study demonstrates that ASC-Exos effectively promote peripheral nerve regeneration via optimizing SC function and thereby represent a novel therapeutic strategy for regenerative medicine and nerve tissue engineering.