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.     

Unchanged rate of axonal regeneration by cyclophosphamide in rat sciatic nerve

The rates of axonal regeneration and initial delay in motor and sensory axons of cyclophosphamide-treated and control rat sciatic nerves after cold injury were determined by using fast axoplasmic transport. The rates in motor and sensory nerves were not significantly different between the two groups. The difference of initial delay in motor nerve was not significant, but in sensory nerve the drug-treated group showed a longer initial delay than the control. These results suggest that the enhancement of motor function recovery by cyclophosphamide is not due directly to an increased rate of axonal regeneration, nor to a decreased initial delay.     

Potentiation of angiogenesis and regeneration by G-CSF after sciatic nerve crush injury

Granulocyte colony-stimulating factor (G-CSF) demonstrates neuroprotective effects through different mechanisms, including mobilization of bone marrow cells. However, the influence of G-CSF-mediated mobilization of bone marrow-derived cells on injured sciatic nerves remains to be elucidated. The administration of G-CSF promoted a short-term functional recovery 7 days after crush injury in sciatic nerves. A double-immunofluorescence study using green fluorescent protein-chimeric mice revealed that bone marrow-derived CD34+ cells were predominantly mobilized and migrated into injured nerves after G-CSF treatment. G-CSF-mediated beneficial effects against sciatic nerve injury were associated with increased CD34+ cell deposition, vascular endothelial growth factor (VEGF) expression, and vascularization/angiogenesis as well as decreased CD68+ cell accumulation. However, cell differentiation and VEGF expression were not demonstrated in deposited cells. The results suggest that the promotion of short-term functional recovery in sciatic nerve crush injury by G-CSF involves a paracrine modulatory effect and a bone marrow-derived CD34+ cell mobilizing effect.     

Increased expression of Gem after rat sciatic nerve injury

Gem belongs to the Rad/Gem/Kir subfamily of Ras-related GTPases, whose expression is induced in several cell types upon activation by extracellular stimuli. Two functions of Gem have been demonstrated, including regulation of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. Because of the essential relationship between actin reorganization and peripheral nerve regeneration, we investigated the spatiotemporal expression of Gem in a rat sciatic nerve crush (SNC) model. After never injury, we observed that Gem had a significant up-regulation from 1 day, peaked at day 5 and then gradually decreased to the normal level. At its peak expression, Gem expressed mainly in Schwann cells (SCs) and macrophages of the distal sciatic nerve segment, but had few colocalization in axons. In addition, the peak expression of Gem was in parallel with PCNA, and numerous SCs expressing Gem were PCNA positive. Thus, all of our findings suggested that Gem may be involved in the pathophysiology of sciatic nerve after SNC.     

In vivo mechanisms of acetylcholine-induced vasodilation in rat sciatic nerve

We examined the importance of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and neurogenic activity in agonist-induced vasodilation and baseline blood flow [i.e., nerve microvascular conductance (NMVC)] in rat sciatic nerve using laser Doppler flowmetry. Agonists were acetylcholine (ACh) and 3-morpholinosydnonimine (SIN-1). Vasodilation occurring despite NO synthase (NOS) and cyclooxygenase inhibition and showing dependence on K(+) channel activity was taken as being mediated by EDHF. NOS and cyclooxygenase inhibition with N(omega)-nitro-L-arginine (L-NNA) + indomethacin (Indo) revealed two phases of ACh-induced vasodilation: an initial, transient L-NNA + Indo-resistant vasodilation, peaking at 23 +/- 6 s and lasting 145 +/- 69 s, followed by sustained L-NNA + Indo-sensitive vasodilation. L-NNA alone did not affect sustained ACh-induced vasodilation but decreased baseline NMVC by 55%. In the presence of L-NNA + Indo, the K(+) channel blocker tetraethylammonium (TEA) inhibited transient ACh-induced vasodilation by 58% and reduced baseline NMVC by 25%. SIN-1-induced vasodilation increased fourfold in the presence of L-NNA, whereas the specific guanylyl cyclase inhibitor 1H-(1, 2, 4)oxadiazolo(4,3-alpha)quinoxalin-1-one abolished it. However, in homogenates of rat sciatic nerve, SIN-1-stimulated soluble guanylyl cyclase (sGC) activity was unaffected by L-NNA. TTX affected neither SIN-1- nor ACh-induced vasodilation. In conclusion, ACh-induced vasodilation consisted of two components, the first partially mediated by EDHF and the second by a vasodilatory prostanoid + NO. Baseline NMVC was dependent on NO and EDHF. Although L-NNA enhanced SIN-1-induced vasodilation, it had no effect on sGC-activity.     

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.     

NGF receptor-mediated reduction in axonal NGF uptake and retrograde transport following sciatic nerve injury and during regeneration.

Injury to the rat sciatic nerve leads to the induction of nerve growth factor (NGF) receptors on the denervated Schwann cells and their disappearance on the regenerating axons of the axotomized, normally NGF-sensitive sensory and sympathetic neurons. This disappearance in the axonal expression and retrograde transport of NGF receptors is associated with a similarly dramatic reduction in the axonal uptake and retrograde transport of NGF following axotomy and during regeneration. In view of the massive NGF synthesis occurring in the injured nerve, these results suggest that, while sensory and sympathetic neurons are the primary targets of NGF in the normal peripheral nervous system, the denervated Schwann cells may become its primary target in the aftermath of nerve injury.     

Transplantation of adipose derived stem cells for peripheral nerve regeneration in sciatic nerve defects of the rat

Tissue engineering approaches for promoting the repair of peripheral nerve injuries have focused on cell-based therapies involving Adipose-derived stem cells (ASCs). The authors evaluated the effects of undifferentiated ASCs and of neurally differentiated ASCs on the regenerating abilities of peripheral nerves. We hope that this would demonstrate the feasibility of using adipose derived stem cells for peripheral nerve regeneration and provide clues regarding the use of adipose- derived stem cells. ASCs were isolated and cultured. Then the cells were cultured with neuronal induction agents for neural differentiation. ASCs and neurally differentiated ASCs were transplanted into sciatic nerve defects. After 12 weeks, the number and diameter of the myelinated fibers were measured and nerve conduction study was done. The extent of regeneration of myelinated fibers in the neurally differentiated ASCs transplanted group was greater than that in the ASCs transplanted group or the control group. However, thickness of myelin sheath and diameter of nerve fibers in the ASCs transplanted group were greater than those in the neutrally differentiated ASCs transplanted group or the control group. Nerve conduction study showed good recovery in the neurally differentiated ASCs transplanted groups. Muscles can atrophy and contract if denervation has started. It would be difficult to recover muscle function even if the nerve was reinnervated. Therefore, although neurally differentiated ASCs were found to have a greater functional effect than non-differentiated ASCs, time constraint is important when considering a method of ASCs transplantation.     

Proteomics of the injured rat sciatic nerve reveals protein expression dynamics during regeneration

Using proteomics, we investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the approximately 1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, alpha-crystallin B, alpha-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

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     

Effects of noradrenaline and propranolol on the regeneration neuroma of the rat sciatic nerve

The effects of noradrenaline (NA) and propranolol (PR) on the regeneration neuroma (RN) of the sciatic nerve were studied in albino rats. It was shown that adrenotropic compounds can substantially influence formation of the RN. Noradrenaline decreased, while PR increased the contents of fibroblasts and neuromyelocytes in the regenerating structures. At the same time, NA increased and PR decreased the specific length of regenerating nerve fibers. The dose—response plots were of an S-like shape, which allowed us to assume specificity of the observed reactions. The reactions of different RN tissue components to the effects of NA and PR differ from each other both quantitatively and qualitatively, while their intensity depends on the phase of the recovery process.     

Effect of a high-intensity static magnetic field on sciatic nerve regeneration in the rat

The effect of a high-intensity static magnetic field on peripheral nerve regeneration is evaluated in rat sciatic nerve. Forty-four rats underwent sciatic nerve repair using polyethylene nerve guides. Postoperatively, the animals were exposed to a 1-tesla magnetic field for 12 hours per day for 4 weeks with appropriate controls. Our results demonstrate that a 1-tesla static magnetic field has no statistically significant effect on nerve regeneration as determined by myelinated axon counts and electrophysiologic studies. Also, the specific orientation of the sciatic nerve with respect to the magnetic field has no influence on axonal growth or nerve conduction. Periods of restraint of 12 hours per day for 4 weeks significantly inhibit weight gain but have no effect on peripheral nerve regeneration.     

Detection of sodium channel distribution in rat sciatic nerve following lysophosphatidylcholine-induced demyelination

Summary In vivo application of lysophosphatidylcholine (LPC) to rat sciatic nerve induces impaired hind leg movement within 2 days which is recovered by 6 days. Segmental demyelination was seen at 2 days after LPC application, and remyelination had barely started in a few axons by 6 days. Using sodium channel-specific monoclonal antibodies and immunofluorescence microscopy, we observed altered distribution of sodium channels in demyelinated axons. Bright fluorescent labeling was found along the segmentally demyelinated axolemma at 6 days in contrast to the dim staining of the demyelinated nerve found at 2 days. In addition, radioimmunoassays detected an elevated number of antibody binding sites on sciatic nerve trunk from the sixth day. Our data provide the immunocytochemical evidence for the assumption that recruitment of sodium channels into demyelinated axolemma contributes to the recovery of function following axon demyelination by LPC.     

Simvastatin对大鼠坐骨神经crush损伤修复作用研究

目的：探讨他汀类（statins）药物Simvastatin在大鼠坐骨神经损伤修复中的作用及可能的作用机制。方法：制作SD大鼠标准坐骨神经钳夹损伤（crush）模型后,分别予Simvastatin和溶媒对照干预2周。手术前后不同时间点进行趾展功能指数测定、神经电生理学、血脂水平、血清IL-6检测和组织学评价。结果：Simvastatin干预组与对照组比较,趾展功能指数在术后5d和8d显著增大（P〈0．05）,足趾展开速度快;2周肌肉复合动作电位幅度高,4周神经传导速度快;组织学显示有髓神经纤维数量多,髓鞘厚,排列相对整齐。各组手术前血脂水平无差异,手术后2周均有不同程度的降低,但Simvastatin干预组总胆固醇降低程度最轻,与对照组比较有显著差异（P〈0．05）;Simvastatin干预组手术后5d,血清IL-6水平明显低于对照组（P〈0．05）。结论：本研究发现,Simvastatin可能通过抑制免疫炎症反应,维持神经损伤后胆固醇的平衡,促进大鼠坐骨神经损伤的修复和再生。     

Neuroprotective effect of sonic hedgehog up-regulated in Schwann cells following sciatic nerve injury

The physiological roles of sonic hedgehog (Shh) have been intensively characterized in development of various organs. However, their functions in adult tissues have not been fully elucidated. We investigated the expression and the potential function of Shh in crush-injured adult rat sciatic nerves. The Shh expression was up-regulated in Schwann cells adjacent to the injured site. The time-course analyses of various neurotrophic factors revealed the up-regulation of Shh mRNA followed by that of brain-derived neurotrophic factor (BDNF) mRNA. The continuous administration of cyclopamine, a hedgehog signal inhibitor, to the injured site suppressed the increase of BDNF expression and deteriorated the survival of motor neurons in lumbar spinal cord. Treatment of exogenous Shh in cultured Schwann cells enhanced the BDNF expression. The BDNF promoter activity (exon I and II) was increased in IMS32 cells co-transfected with Shh and its receptor Smoothened. These findings imply that the up-regulated expression of Shh in Schwann cells may play an important role in injured motor neurons through the induction of BDNF.     

利用深度测序技术检测玉米根系和叶片中已知的microRNAs

microRNA(miRNA)是一类具有20～24nt核苷酸长度的非蛋白质编码的内源小分子RNA,它在植物生长发育和逆境胁迫响应等过程中发挥着重要作用。文章利用基于Illumina/Solexa原理的小分子RNA深度测序技术,结合生物信息学的方法对玉米根系和叶片中已知miRNA的类型、丰度及靶基因进行了分析。研究发现,在根系中共检测到92个已知的miRNA,分别属于18个miRNA家族,其表达丰度在1～105943之间;在叶片中,共发现86个已知的miRNA,分别属于17个miRNA家族,其表达丰度在1～85973之间。靶基因预测结果表明,根系中的18个miRNA家族共靶向54个蛋白,进一步的功能预测发现,这些基因涉及了转录调控、物质能量代谢、电子传递、胁迫响应和信号转导等过程。以上研究结果表明,就已知的miRNA而言,无论是miRNA的类型还是表达丰度,在玉米根系和叶片中都存在较大差异。     

Focal regeneration in the rat myocardium following cold injury

Summary The sequential cytological events in the myocardium of the rat were followed for 3 weeks after cold injury by light and electron microscopy. The traumatized area was initially filled with leukocytes and undifferentiated mononucleated cells and subsequently mainly with fibroblasts surrounded by collagen fibers. However, in the margins of the necrotic area repair processes of damaged myocardial cells and probably also the appearance of newly formed cells were evident. The ultrastructural features of these cells were characterized by clusters of ribosomes, numerous mitochondria that were dispersed in the cytoplasm and formation of junctional complexes and transverse tubular systems. Fibrillogenesis was also clearly evident in these cardiomyocytes. The myofibrillar material was initially dispersed in the cytoplasm and associated with clusters of ribosomes and thereafter with presumptive Z-bands and intercalated discs. The myofibrils became further organized in the shape and orientation of those of mature cells two to three weeks after injury. It is concluded that following cold injury regeneration in the mammalian myocardium takes place but is limited to the perinecrotic area. The process resembles the sequential cytological events which occur in cardiomyocytes during embryonic and postnatal development of the ventricular myocardium.