Post-injury regeneration in rat sciatic nerve facilitated by neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.

Amniotic fluid mesenchymal stem cells have the ability to secrete neurotrophic factors that are able to promote neuron survival in vitro. The purpose of this study was to evaluate the effects of neurotrophic factors secreted by rat amniotic fluid mesenchymal stem cells on regeneration of sciatic nerve after crush injury. Fifty Sprague-Dawley rats weighing 250-300 g were used. The left sciatic nerve was crushed with a vessel clamp. Rat amniotic fluid mesenchymal stem cells embedded in fibrin glue were delivered to the injured nerve. Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry were used to detect neurotrophic factors secreted by the amniotic fluid mesenchymal stem cells. Nerve regeneration was assessed by motor function, electrophysiology, histology, and immunocytochemistry studies. Positive CD29/44, and negative CD11b/45, as well as high levels of expression of brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor, ciliary neurotrophic factor (CNTF), nerve growth factor, and neurotrophin-3 (NT-3) were demonstrated in amniotic fluid mesenchymal stem cells. Motor function recovery, the compound muscle action potential, and nerve conduction latency showed significant improvement in rats treated with amniotic fluid mesenchymal stem cells. ELISA measurement in retrieved nerves displayed statistically significant elevation of CNTF and NT-3. The immunocytochemical studies demonstrated positive staining for NT-3 and CNTF in transplanted cells. The histology and immunocytochemistry studies revealed less fibrosis and a high level of expression of S-100 and glial fibrillary acid protein at the crush site. Rat amniotic fluid mesenchymal stem cells may facilitate regeneration in the sciatic nerve after crush injury. The increased nerve regeneration found in this study may be due to the neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.     

Exenatide promotes regeneration of injured rat sciatic nerve

Damage to peripheral nerves results in partial or complete dysfunction. After peripheral nerve injuries, a full functional recovery usually cannot be achieved despite the standard surgical repairs. Neurotrophic factors and growth factors stimulate axonal growth and support the viability of nerve cells. The objective of this study is to investigate the neurotrophic effect of exenatide(glucagon like peptide-1 analog) in a rat sciatic nerve neurotmesis model. We injected 10 μg/d exenatide for 12 weeks in the experimental group(n = 12) and 0.1 m L/d saline for 12 weeks in the control group(n = 12). We evaluated nerve regeneration by conducting electrophysiological and motor functional tests. Histological changes were evaluated at weeks 1, 3, 6, and 9. Nerve regeneration was monitored using stereomicroscopy. The electrophysiological and motor functions in rats treated with exenatide were improved at 12 weeks after surgery. Histological examination revealed a significant increase in the number of axons in injured sciatic nerve following exenatide treatment confirmed by stereomicroscopy. In an experimentally induced neurotmesis model in rats, exenatide had a positive effect on nerve regeneration evidenced by electromyography, functional motor tests, histological and stereomicroscopic findings.     

Combination of olfactory ensheathing cells and human umbilical cord mesenchymal stem cell-derived exosomes promotes sciatic nerve regeneration

Olfactory ensheathing cells(OECs)are promising seed cells for nerve regeneration.However,their application is limited by the hypoxic environment usually present at the site of injury.Exosomes derived from human umbilical cord mesenchymal stem cells have the potential to regulate the pathological processes that occur in response to hypoxia.The ability of OECs to migrate is unknown,especially in hypoxic conditions,and the effect of OECs combined with exosomes on peripheral nerve repair is not clear.Better understanding of these issues will enable the potential of OECs for the treatment of nerve injury to be addressed.In this study,OECs were acquired from the olfactory bulb of Sprague Dawley rats.Human umbilical cord mesenchymal stem cell-derived exosomes(0–400μg/mL)were cultured with OECs for 12–48 hours.After culture with 400μg/mL exosomes for 24 hours,the viability and proliferation of OECs were significantly increased.We observed changes to OECs subjected to hypoxia for 24 hours and treatment with exosomes.Exosomes significantly promoted the survival and migration of OECs in hypoxic conditions,and effectively increased brain-derived neurotrophic factor gene expression,protein levels and secretion.Finally,using a 12 mm left sciatic nerve defect rat model,we confirmed that OECs and exosomes can synergistically promote motor and sensory function of the injured sciatic nerve.These findings show that application of OECs and exosomes can promote nerve regeneration and functional recovery.This study was approved by the Institutional Ethical Committee of the Air Force Medical University,China(approval No.IACUC-20181004)on October 7,2018;and collection and use of human umbilical cord specimens was approved by the Ethics Committee of the Linyi People’s Hospital,China(approval No.30054)on May 20,2019.     

脐带间充质干细胞与许旺细胞共移植修复大鼠坐骨神经缺损

Previous research has demonstrated that cotransplantation of umbilical cord mesenchymal stem cells (UCMSCs) and Schwann cells (SCs) can repair spinal nerve injury, but few studies have investigated their use in peripheral nerve regeneration. In the present study, we cotransplanted UCMSCs and SCs to repair 5-mm left sciatic nerve defects in rats, and compared the effects of UCMSCs + SCs transplantation with UCMSCs or SCs transplantation alone. After UCMSCs + SCs transplantation, nerve conduction velocity of the left sciatic nerve and gait were both improved. Retrograde tracing analysis demonstrated that the mean count of fluorogold-labeled neurons, as well as the mean axon count and axon density, were significantly greater in the left sciatic nerve after UCMSCs + SCs transplantation, compared with UCMSCs or SCs transplantation alone. Improvements in conduction velocity and increased sheath thickness in the left sciatic nerve were similar after UCMSCs transplantation and UCMSCs + SCs transplantation. These findings suggest that UCMSCs transplantation can promote the repair of sciatic nerve defects to some extent, but that combined UCMSCs + SCs transplantation has a significantly greater regenerative effect.     

Electroacupuncture and moxibustion promote regeneration of injured sciatic nerve through Schwann cell proliferation and nerve growth factor secretion

Using electroacupuncture and moxibustion to treat peripheral nerve injury is highly efficient with low side effects. However, the electroacupuncture-and moxibustion-based mechanisms underlying nerve repair are still unclear. Here, in vivo and in vitro experiments uncovered one mechanism through which electroacupuncture and moxibustion affect regeneration after peripheral nerve injury. We first established rat models of sciatic nerve injury using neurotomy. Rats were treated with electroacupuncture or moxibustion at acupoints Huantiao(GB30) and Zusanli(ST36). Each treatment lasted 15 minutes, and treatments were given six times a week for 4 consecutive weeks. Behavioral testing was used to determine the sciatic functional index. We used electrophysiological detection to measure sciatic nerve conduction velocity and performed hematoxylin-eosin staining to determine any changes in the gastrocnemius muscle. We used immunohistochemistry to observe changes in the expression of S100—a specific marker for Schwann cells—and an enzyme-linked immunosorbent assay to detect serum level of nerve growth factor. Results showed that compared with the model-only group, sciatic functional index, recovery rate of conduction velocity, diameter recovery of the gastrocnemius muscle fibers, number of S100-immunoreactive cells, and level of nerve growth factor were greater in the electroacupuncture and moxibustion groups. The efficacy did not differ between treatment groups. The serum from treated rats was collected and used to stimulate Schwann cells cultured in vitro. Results showed that the viability of Schwann cells was much higher in the treatment groups than in the model group at 3 and 5 days after treatment. These findings indicate that electroacupuncture and moxibustion promoted nerve regeneration and functional recovery; its mechanism might be associated with the enhancement of Schwann cell proliferation and upregulation of nerve growth factor.     

Enhanced functional recovery from sciatic nerve crush injury through a combined treatment of cold-water swimming and mesenchymal stem cell transplantation

Abstract

Objective:

Although regimens of stem cell implantation can elicit functional recovery following peripheral nerve injury, the degree of outcome is still limited. This study evaluated the synergistic effects of cold-water swimming (CWS) and mesenchymal stem cell (MSC) transplantation on functional recovery of crushed sciatic nerve in rats.

Method:

Forty Sprague-Dawley rats that had their sciatic nerve crushed during surgery were randomly divided into four groups: MSCCWS group, treated with combination of MSC and CWS; MSC group, treated with MSC alone; CWS group, treated with CWS alone; and non-treated group, without any treatments. The sciatic function index (SFI), vertical activity (VA), ankle activity (AA) and electrophysiological study were examined before, immediately after surgery, after the treatment and after 4?weeks from treatment. Morphological and S100 immunohistochemical studies were also performed.

Results:

The MSCCWS group showed a greater improvement in SFI, VA, AA, peak amplitudes and onset latencies of compound muscle action potential (CMAP) in sciatic nerve and infiltration of immune cells with significant difference from the MSC, CWS and non-treated groups (P?<?0.05).

Conclusions:

MSC transplantation combined with CWS could achieve better results in functional recovery than a single treatment of MSC alone or CWS alone in nerve crush injury.     

Effects of FK506 on regeneration and macrophages in injured rat sciatic nerve

Effects of FK506 [5.0 mg/kg body weight (BW), subcutaneous, daily] on nerve regeneration and presence of macrophages in lesioned rat sciatic nerves were studied. Models of autologous nerve graft or a nerve crush lesion were used and regeneration was evaluated by immunocytochemistry (also used to detect ED1/ED2 macrophages) and sensory pinch reflex test, respectively. Treatment with FK506 did not increase regeneration distance or regeneration rate in the autologous nerve grafts. However, regeneration distances after nerve crush were significantly longer following treatment with FK506. The number of macrophages (ED1/ED2) in nerve grafts increased over time, but treatment with FK506 had limited effects only in the presence of ED2 macrophages. Present and previously published studies may imply that there is a time-related and type-of-injury-related profile of FK506's pro-regenerative effect.     

Bone marrow-derived mesenchymal stem cells versus adipose-derived mesenchymal stem cells for peripheral nerve regeneration

Studies have confirmed that bone marrow-derived mesenchymal stem cells(MSCs) can be used for treatment of several nervous system diseases. However, isolation of bone marrow-derived MSCs(BMSCs) is an invasive and painful process and the yield is very low. Therefore, there is a need to search for other alterative stem cell sources. Adipose-derived MSCs(ADSCs) have phenotypic and gene expression profiles similar to those of BMSCs. The production of ADSCs is greater than that of BMSCs, and ADSCs proliferate faster than BMSCs. To compare the effects of venous grafts containing BMSCs or ADSCs on sciatic nerve injury, in this study, rats were randomly divided into four groups: sham(only sciatic nerve exposed), Matrigel(MG; sciatic nerve injury + intravenous transplantation of MG vehicle), ADSCs(sciatic nerve injury + intravenous MG containing ADSCs), and BMSCs(sciatic nerve injury + intravenous MG containing BMSCs) groups. Sciatic functional index was calculated to evaluate the function of injured sciatic nerve. Morphologic characteristics of nerves distal to the lesion were observed by toluidine blue staining. Spinal motor neurons labeled with Fluoro-Gold were quantitatively assessed. Compared with sham-operated rats, sciatic functional index was lower, the density of small-diameter fibers was significantly increased, and the number of motor neurons significantly decreased in rats with sciatic nerve injury. Neither ADSCs nor BMSCs significantly improved the sciatic nerve function of rats with sciatic nerve injury, increased fiber density, fiber diameters, axonal diameters, myelin sheath thickness, and G ratios(axonal diameter/fiber diameter ratios) in the sciatic nerve distal to the lesion site. There was no significant difference in the number of spinal motor neurons among ADSCs, BMSCs and MG groups. These results suggest that neither BMSCs nor ADSCs provide satisfactory results for peripheral nerve repair when using MG as the conductor for engraftment.     

Unilateral sciatic nerve injury stimulates contralateral nerve regeneration.

Axonal outgrowth in tissue cultures was measured to determine whether unilateral peripheral nerve injuries affect contralateral nerve regeneration. The right sciatic nerves of young male Wistar rats were cut at mid-thigh level. Sham operation as a control was limited to the exposure of the nerve without cutting. At day 6 post-surgery, bilateral L5 dorsal root ganglia (DRG) with attached nerve stumps were resected and cultured. Axonal outgrowth from the nerve stump was measured in situ. The contralateral preparations showed longer outgrowths than controls. Therefore the conditioning effect was not merely restricted to the ipsilateral neurons but also affected undamaged sensory neurons of the contralaretal DRG.     

Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents

Following a peripheral nerve injury, brain-derived neurotrophic factor (BDNF) and the p75 neurotrophin receptor are upregulated in Schwann cells of the Wallerian degenerating nerves. However, it is not known whether the endogenous BDNF is critical for the functions of Schwann cells and regeneration of injured nerve. Treatment with BDNF antibody was shown to retard the length of the regenerated nerve from injury site by 24%. Histological and ultrastructural examination showed that the number and density of myelinated axons in the distal side of the lesion in the antibody-treated mice was reduced by 83%. In the BDNF antibody-treated animals, there were only distorted and disorganized myelinated fibres in the injured nerve where abnormal Schwann cells and phagocytes were present. As a result of nerve degeneration in BDNF antibody-treated animals, subcellular organelles, such as mitochondria, disappeared or were disorganized and the laminal layers of the myelin sheath were loosened, separated or collapsed. Our in situ hybridization revealed that BDNF mRNA was expressed in Schwann cells in the distal segment of lesioned nerve and in the denervated muscle fibres. These results indicate that Schwann cells and muscle fibres may contribute to the sources of BDNF during regeneration and that the deprivation of endogenous BDNF results in an impairment in regeneration and myelination of regenerating axons. It is concluded that endogenous BDNF is required for peripheral nerve regeneration and remyelination after injury.     

靶肌肉注射人间充质干细胞对坐骨神经损伤保护性作用的电生理研究

目的探讨电生理在大鼠失神经支配的骨骼肌中移植间充质干细胞对坐骨神经损伤后修复中的作用。方法体外分离培养人间充质干细胞,经鉴定标记后备用。将36只SD大鼠随机分为肌肉注射干细胞组(实验组)、肌肉注射生理盐水组(对照组),每组18只,建立大鼠坐骨神经损伤模型,术后3d将干细胞和等量生理盐水注射到损伤侧坐骨神经支配的骨骼肌中。于移植后3、7、14、21、28、60d,观察坐骨神经功能指数(SFI)、腓肠肌肌电图(EMG)、运动神经传导速度(MCV),肌肉复合动作电位幅值(CMAP)检查。结果腓肠肌EMG实验组自发电位减少和动作电位出现时间均早于对照组。坐骨神经MCV和CMAP波幅呈恢复趋势。移植后:21d开始,实验组明显快于对照组(P<0.05),60d时,传导速度两组无显著性(P>0.05),波幅有显著性差异(P<0.05)。结论肌肉注射人间充质干细胞对坐骨神经损伤具有促修复作用。     

Comparison of viscoelasticity between normal human sciatic nerve and amniotic membrane

Sciatic nerve tissue was obtained from the gluteus maximus muscle segment of normal human cadavers and amniotic membrane tissue was obtained from healthy human puerperant placentas.Both tissues were analyzed for their stress relaxation and creep properties to determine suitability for transplantation applications.Human amniotic membrane and sciatic nerve tissues had similar tendencies for stress relaxation and creep properties.The stress value of the amniotic membrane stress relaxation group decreased to a greater extent compared with the sciatic nerve stress relaxation group.Similarly,the stress value of the amniotic membrane creep group increased to a greater extent compared with the sciatic nerve creep group.The stress relaxation curve for human amniotic membrane and sciatic nerve showed a logarithm correlation,while the creep curve showed an exponential correlation.These data indicate that amniotic membrane tissue has better stress relaxation and creep properties compared with sciatic nerve tissue.     

Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection

The regeneration in the peripheral nervous system is often incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity. Guide tubes of various types, filled with Schwann cells, stem cells, or nerve growth factors are attractive as an alternative therapy to nerve grafts. In this study, we evaluated whether skin-derived stem cells (SDSCs) can improve peripheral nerve regeneration after transplantation into nerve guides. We compared peripheral nerve regeneration in adult rats with sciatic nerve gaps of 16 mm after autologous transplantation of GFP-labeled SDSCs into two different types of guides: a synthetic guide, obtained by dip coating with a L-lactide and trimethylene carbonate (PLA-TMC) copolymer and a collagen-based guide. The sciatic function index and the recovery rates of the compound muscle action potential were significantly higher in the animals that received SDSCs transplantation, in particular, into the collagen guide, compared to the control guides filled only with PBS. For these guides the morphological and immunohistochemical analysis demonstrated an increased number of myelinated axons expressing S100 and Neurofilament 70, suggesting the presence of regenerating nerve fibers along the gap. GFP positive cells were found around regenerating nerve fibers and few of them were positive for the expression of glial markers as S-100 and glial fibrillary acidic protein. RT-PCR analysis confirmed the expression of S100 and myelin basic protein in the animals treated with the collagen guide filled with SDSCs. These data support the hypothesis that SDSCs could represent a tool for future cell therapy applications in peripheral nerve regeneration.     

无细胞神经支架复合骨髓间充质干细胞构建组织工程人工神经修复坐骨神经缺损*☆

背景：作者已经成功制备了无细胞神经移植物,并且复合骨髓间充质干细胞构建组织工程人工神经桥接大鼠坐骨神经缺损。 目的：无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复大鼠坐骨神经缺损后运动功能的恢复。 方法：成年雄性SD大鼠构建大鼠坐骨神经15 mm缺损模型,分别应用组织工程人工神经、组织工程神经支架或自行神经桥接坐骨神经缺损。桥接后20周再生神经电生理学测定,手术侧胫骨前肌湿质量、腓肠肌组织学及透视电镜分析。 结果与结论：桥接20周后,组织工程人工神经与自体神经移植组胫骨前肌湿质量比较,差异无显著性意义(P > 0.05),神经干传导速度为(30.56±2.15)m/s。结果提示,无细胞神经移植物复合骨髓间充质干细胞构建的组织工程人工神经桥接大鼠坐骨神经缺损后,可以促进再生神经运动功能的恢复。     

Autologous nerve anastomosis versus human amniotic membrane anastomosisA rheological comparison following simulated sciatic nerve injury

The sciatic nerve is biological viscoelastic solid,with stress relaxation and creep characteristics.In this study,a comparative analysis of the stress relaxation and creep characteristics of the sciatic nerve was conducted after simulating sciatic nerve injury and anastomosing with autologous nerve or human amniotic membrane.The results demonstrate that,at the 7 200-second time point,both stress reduction and strain increase in the human amniotic membrane anastomosis group were significantly greater than in...     

Human umbilical cord blood-derived mesenchymal stem cells promote regeneration of crush-injured rat sciatic nerves

Several studies have demonstrated that human umbilical cord blood-derived mesenchymal stem cells can promote neural regeneration following brain injury. However, the therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells in guiding peripheral nerve regeneration remain poorly understood. This study was designed to investigate the effects of human umbilical cord blood-derived mesenchymal stem cells on neural regeneration using a rat sciatic nerve crush injury model. Human umbilical cord blood-derived mesenchymal stem cells (1 × 10 6 ) or a PBS control were injected into the crush-injured segment of the sciatic nerve. Four weeks after cell injection, brain-derived neurotrophic factor and tyrosine kinase receptor B mRNA expression at the lesion site was increased in comparison to control. Furthermore, sciatic function index, Fluoro Gold-labeled neuron counts and axon density were also significantly increased when compared with control. Our results indicate that human umbilical cord blood-derived mesenchymal stem cells promote the functional recovery of crush-injured sciatic nerves.     

Transplantation of mesenchymal stem cells from human umbilical cord versus human umbilical cord blood for peripheral nerve regeneration

BACKGROUND: Mesenchymal stem cells (MSCs) appear to be a good alternative to Schwann cells in the treatment of peripheral nerve injury. Fetal stem cells, like umbilical cord blood (UCB) and umbilical cord (UC) stem cells, have several advantages over adult stem cells.OBJECTIVE: To assess the effects of UC-derived MSCs (UCMSCs) and UCB-derived MSCs (UCBMSCs) in repair of sciatic nerve defects. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the laboratory of Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, from July to December 2009. MATERIALS: UCMSCs were provided by the Research Institute of Biotechnology, Dongguk University. UCBMSCs were provided by the Laboratory of Stem Cells and Tumor Biology, College of Veterinary Medicine, Seoul National University. Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco-BRL, USA. METHODS: Seven-week-old Sprague-Dawley rats were randomly and evenly divided into three groups: DMEM, UCBMSCs, and UCMSCs. A 10-mm defect in the left sciatic nerve was constructed in all rats. DMEM (15 μL) containing 1 × 106 UCBMSCs or UCMSCs was injected into the gap between nerve stumps, with the surrounding epineurium as a natural conduit. For the DMEM group, simple DMEM was injected. MAIN OUTCOME MEASURES: At 7 weeks after sciatic nerve dissection, dorsal root ganglia neurons were labeled by fluorogold retrograde labeling. At 8 weeks, electrophysiology and histomorphometry were performed. At 2, 4, 6, and 8 weeks after surgery, sciatic nerve function was evaluated using gait analysis.RESULTS: The UCBMSCs group and the UCMSCs group exhibited similar sciatic nerve function and electrophysiological indices, which were better than the DMEM group, as measured by gait analysis (P < 0.05). Fluorogold retrograde labeling of sciatic nerve revealed that the UCBMSCs group demonstrated a higher number of labeled neurons; however, the differences were not significant. Histomorphometric indices were similar in the UCBMSCs and UCMSCs groups, and total axon counts, particularly axon density (P < 0.05), were significantly greater in the UCBMSCs and UCMSCs groups than in the DMEM group. CONCLUSION: Transplanting either UCBMSCs or UCMSCs into axotomized sciatic nerves could accelerate and promote sciatic nerve regeneration over 8 weeks. Both treatments had similar effects on nerve regeneration.     

Extracellular matrix from human umbilical cord-derived mesenchymal stem cells as a scaffold for peripheral nerve regeneration

The extracellular matrix, which includes collagens, laminin, or fibronectin, plays an important role in peripheral nerve regeneration. Recently, a Schwann cell-derived extracellular matrix with classical biomaterial was used to mimic the neural niche. However, extensive clinical use of Schwann cells remains limited because of the limited origin, loss of an autologous nerve, and extended in vitro culture times. In the present study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs), which are easily accessible and more proliferative than Schwann cells, were used to prepare an extracellular matrix. We identiifed the morphology and function of hUCMSCs and investi-gated their effect on peripheral nerve regeneration. Compared with a non-coated dish tissue culture, the hUCMSC-derived extracellular matrix enhanced Schwann cell proliferation, upregulated gene and protein expression levels of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor in Schwann cells, and enhanced neurite outgrowth from dorsal root ganglion neurons. These ifndings suggest that the hUCMSC-derived extracellular matrix promotes peripheral nerve repair and can be used as a basis for the rational design of engineered neural niches.