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In JoVE (1)
Other Publications (11)
- The Journal of Bone and Joint Surgery. British Volume
- Scandinavian Journal of Plastic and Reconstructive Surgery and Hand Surgery / Nordisk Plastikkirurgisk Forening [and] Nordisk Klubb for Handkirurgi
- Scandinavian Journal of Plastic and Reconstructive Surgery and Hand Surgery / Nordisk Plastikkirurgisk Forening [and] Nordisk Klubb for Handkirurgi
- Plastic and Reconstructive Surgery
- The Journal of Hand Surgery, European Volume
- Proceedings of the National Academy of Sciences of the United States of America
- Hand Surgery : an International Journal Devoted to Hand and Upper Limb Surgery and Related Research : Journal of the Asia-Pacific Federation of Societies for Surgery of the Hand
- Genes & Development
- Nature Communications
- Trends in Neurosciences
- Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society
Articles by Saijilafu in JoVE
JoVE Neuroscience
Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons
1Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, 2Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine
An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.
Other articles by Saijilafu on PubMed
Effect of Progesterone on Recovery from Nerve Injury During Leg Lengthening in Rats
We investigated the effect of progesterone on the nerve during lengthening of the limb in rats. The sciatic nerves of rats were elongated by leg lengthening for ten days at 3 mm per day. On alternate days between the day after the operation and nerve dissection, the progesterone-treated group received subcutaneous injections of 1 mg progesterone in sesame oil and the control group received oil only. On the fifth, tenth and 17th day, the sciatic nerves were excised at the midpoint of the femur and the mRNA expression level of myelin protein P0 was analysed by quantitative real time polymerase chain reaction. On day 52 nodal length was examined by electron microscopy, followed by an examination of the compound muscle action potential (C-MAP) amplitude and the motor conduction velocity (MCV) of the tibial nerve on days 17 and 52. The P0 (a major myelin glycoprotein) mRNA expression level in the progesterone-treated group increased by 46.6% and 38.7% on days five and ten, respectively. On day 52, the nodal length in the progesterone-treated group was smaller than that in the control group, and the MCV of the progesterone-treated group had been restored to normal. Progesterone might accelerate the restoration of demyelination caused by nerve elongation by activating myelin synthesis.
Repair of Peripheral Nerve Defect by Direct Gradual Lengthening of the Distal Nerve Stump in Rats: Cellular Reaction
We investigated the effects of direct gradual lengthening of the distal stump of a peripheral nerve and subsequent nerve regeneration in rats. A segment 10 mm long was resected from rat sciatic nerve. The distal nerve stump was fixed to a ring and pulled directly at a rate of 1 mm/day using an original external nerve distraction device. After distraction for 10, 15, and 20 days, the lengthened nerves were evaluated macroscopically and immunocytochemically. At day 20, the mean (SD) distances from the ring to the 3 mm and 6 mm distal part, which were marked with sutures on the epineurium, were 7 (0.5) mm and 12.1 (0.5) mm, respectively, and the number of Schwann cells in the lengthening group had increased to twice that of control group. The distal stump of a peripheral nerve including the epineurium, endoneurium, and proliferation of Schwann cells can be lengthened directly. This method also made it possible to lengthen the nerve stump longitudinally and to control both the rate and distance. We think that this method may be used in the treatment of peripheral nerve injury.
Repair of Peripheral Nerve Defect by Direct Gradual Lengthening of the Distal Nerve Stump in Rats: Effect on Nerve Regeneration
We investigated the use of direct lengthening of the distal stump of a peripheral nerve to compare the results of nerve regeneration using the direct lengthening method with simple end-to-end suture and autografting in rats. A segment 10 mm long was resected from the rat sciatic nerve (n=18 in each group). The distal nerve stump was fixed to a ring and pulled at a rate of 1 mm/day for 20 days using an original external nerve distraction device. The results showed that the degree of nerve regeneration in the lengthened group was superior to that of the grafted group electrophysiologically and histologically, but there were no significant differences between the lengthened and end-to-end suture groups. We conclude that direct lengthening of the distal stump of a peripheral nerve can promote nerve regeneration similar to that observed in a Wallerian degenerated nerve. We think that this technique may be used for the treatment of peripheral nerve injuries.
Repair of the Sciatic Nerve Defect with a Direct Gradual Lengthening of Proximal and Distal Nerve Stumps in Rabbits
The current clinical repair method used for the segmental peripheral nerve defect is autogenous nerve grafting. However, this method has several inherent disadvantages. Therefore, the authors have invented an alternative method for repairing the segmental peripheral nerve defect with a direct gradual lengthening of nerve stumps. In this study, for the clinical application, the authors developed a new external nerve-lengthening device for lengthening peripheral nerve stumps daily without anesthesia.
The Effects of Repetitive Compression on Nerve Conduction and Blood Flow in the Rabbit Sciatic Nerve
The objective of this study was to clarify the effect of repetitive compression on nerve physiology in an experimental rabbit model. We defined 80 mmHg as a compression force which caused temporary disturbance of nerve conduction and blood flow with a brief compression. The following compressions were applied for 30 minutes to rabbit sciatic nerves: continuous compression, low frequency release compression (1 second of release time every 30 seconds) and high frequency release compression (1 second of release time every 10 seconds). Compound nerve action potentials and nerve blood flow were evaluated from the start of compression until 30 minutes after release. Endoneurial microvascular permeability was evaluated with Evans Blue albumin. The repetitive compression groups showed delay in recovery of compound nerve action potentials and blood flow after release, with endoneurial oedema. These findings suggest that repetitive compression may increase the risk of breakdown of the blood nerve barrier.
Engineering Neuronal Growth Cones to Promote Axon Regeneration over Inhibitory Molecules
Neurons in the central nervous system (CNS) fail to regenerate axons after injuries due to the diminished intrinsic axon growth capacity of mature neurons and the hostile extrinsic environment composed of a milieu of inhibitory factors. Recent studies revealed that targeting a particular group of extracellular inhibitory factors is insufficient to trigger long-distance axon regeneration. Instead of antagonizing the growing list of impediments, tackling a common target that mediates axon growth inhibition offers an alternative strategy to promote axon regeneration. Neuronal growth cone, the machinery that derives axon extension, is the final converging target of most, if not all, growth impediments in the CNS. In this study, we aim to promote axon growth by directly targeting the growth cone. Here we report that pharmacological inhibition or genetic silencing of nonmuscle myosin II (NMII) markedly accelerates axon growth over permissive and nonpermissive substrates, including major CNS inhibitors such as chondroitin sulfate proteoglycans and myelin-associated inhibitors. We find that NMII inhibition leads to the reorganization of both actin and microtubules (MTs) in the growth cone, resulting in MT reorganization that allows rapid axon extension over inhibitory substrates. In addition to enhancing axon extension, we show that local blockade of NMII activity in axons is sufficient to trigger axons to grow across the permissive-inhibitory border. Together, our study proposes NMII and growth cone cytoskeletal components as effective targets for promoting axon regeneration.
Functional and Morphological Effects of Indirect Gradual Elongation of Peripheral Nerve: Electrophysiological and Morphological Changes at Different Elongation Rates
We investigated the neuropathy induced by leg lengthening histological evaluation using teased nerve fiber specimens and electrophysiological evaluation. Indirect elongation of the sciatic nerve associated with leg lengthening was performed at 1 and 3 mm/day over 30 mm in rats. Electrophysiological evaluation was performed immediately and 60 days after the end of elongation, teased nerve fiber specimens were prepared, and the mean axonal diameter was calculated. The electrophysiological results were more wrong, and the recovery was poorer, in the 3-mm than in the 1-mm group. In the 1-mm group, the nerve conduction velocity (NCV) and the duration of the compound nerve action potential (C-NAP) recovered to a level close to the intact side, but the decrease in the amplitude of the C-NAP persisted. In the teased fiber study, while paranodal demyelination was observed in both groups immediately after elongation, demyelination was decreased in the 1-mm group indicationg recovery compared to the 3-mm group. Paranodal demyelination caused by indirect nerve elongation is considered to have induced electrophysiological disorders. Electrophysiological and morphological damages appeared to be more severe according to elongation speed. The nerve disorder were remained even at 1 mm per day in 60 days.
GSK3 Controls Axon Growth Via CLASP-mediated Regulation of Growth Cone Microtubules
Suppression of glycogen synthase kinase 3 (GSK3) activity in neurons yields pleiotropic outcomes, causing both axon growth promotion and inhibition. Previous studies have suggested that specific GSK3 substrates, such as adenomatous polyposis coli (APC) and collapsin response mediator protein 2 (CRMP2), support axon growth by regulating the stability of axonal microtubules (MTs), but the substrate(s) and mechanisms conveying axon growth inhibition remain elusive. Here we show that CLIP (cytoplasmic linker protein)-associated protein (CLASP), originally identified as a MT plus end-binding protein, displays both plus end-binding and lattice-binding activities in nerve growth cones, and reveal that the two MT-binding activities regulate axon growth in an opposing manner: The lattice-binding activity mediates axon growth inhibition induced by suppression of GSK3 activity via preventing MT protrusion into the growth cone periphery, whereas the plus end-binding property supports axon extension via stabilizing the growing ends of axonal MTs. We propose a model in which CLASP transduces GSK3 activity levels to differentially control axon growth by coordinating the stability and configuration of growth cone MTs.
Genetic Dissection of Axon Regeneration Via in Vivo Electroporation of Adult Mouse Sensory Neurons
Manipulating gene expression in vivo specifically in neurons with precise spatiotemporal control is important to study the function of genes or pathways in the nervous system. Although various transgenic approaches or virus-mediated transfection methods are available, they are time consuming and/or lack precise temporal control. Here we introduce an efficient electroporation approach to transfect adult dorsal root ganglion (DRG) neurons in vivo that enables manipulation of gene expression in an acute and precise manner. We have applied this method to manipulate gene expression in three widely used in vivo models of axon injury and regeneration, including dorsal column transection, dorsal root rhizotomy and peripheral axotomy. By electroporating DRGs with small interfering RNAs against c-jun to specifically deplete c-Jun in adult neurons, we provide evidence for the role of c-Jun in regulation of in vivo axon regeneration. This method will serve as a powerful tool to genetically dissect axon regeneration in vivo.
Growing the Growth Cone: Remodeling the Cytoskeleton to Promote Axon Regeneration
Axon growth is driven by the movement of a growth cone, a specialized sensory motile structure located at the tip of a growing neurite. Although stalled retraction bulbs have long been recognized as hallmarks of regeneration failure, mechanisms that control the formation and migration of nerve endings are only beginning to be unraveled. Recent studies point to microtubules as key determinants for such processes, and emerging evidence suggests that regulators of actin and microtubule dynamics in the growth cone might serve as attractive targets for controlling both the speed and trajectory of regenerating axons. This review discusses the potential of and recent progress in direct modulation of the growth cone machinery as a novel strategy to promote axon regeneration in the nervous system after injury.
New Treatment for Peripheral Nerve Defects: Reconstruction of a 2 cm, Monkey Median Nerve Gap by Direct Lengthening of Both Nerve Stumps
We have developed a new treatment for peripheral nerve defects: nerve-lengthening method, and confirmed the efficacy and safety of our method using cynomolgus monkeys. A 20-mm defect in the median nerve of monkey's forearms was repaired through the simultaneous lengthening of both nerve stumps with original nerve-lengthening device. To evaluate nerve regeneration after neurorrhaphy, electrophysiological, histological, and functional recovery were examined and compared to the standard autografting. Nerve conduction velocity, axon maturation, and the result of functional test were superior in the nerve-lengthening method than in the autografting. And there were no adverse events associated with our method. We concluded that this method is practical for clinical application.
