Regeneration Of Injured Axons Research Articles

Mechanism research progress of acupuncture for regulating axon regeneration after stroke.

Stroke brings the pathological changes of brain tissues such as hematoma formation or ischemia and hypoxia, which leads to neuronal death and axon degeneration. Axon regeneration after its injury is mainly dependent on the surrounding microenvironment and the related proteins, including glial scar, myelin associated inhibitory factors, axon guidance molecules, and neurotrophic factors. All of them affect axon growth by regulating the morphology and orientation of growth cones, the synaptic stability, and the proliferation and differentiation of neural stem cells. This article summarizes the mechanism of acupuncture in regulating axon regeneration after stroke. Acupuncture inhibits glial scar formation, alleviates the inhibitory effects of its physical and chemical barriers on axon growth, reverses the inhibitory effects of myelin-related inhibitory factors on axon growth, and adjusts the level of axon guidance molecules to promote the proliferation and differentiation of neural stem cells and the regeneration of injured axons, and up-regulates neurotrophic factors. Eventually, post-stroke nerve injury can be ameliorated.

The role of mechanobiology on the Schwann cell response: A tissue engineering perspective.

Schwann cells (SCs), the glial cells of the peripheral nervous system (PNS), do not only form myelin sheaths thereby insulating the electrical signal propagated by the axons, but also play an essential role in the regeneration of injured axons. SCs are inextricably connected with their extracellular environment and the mechanical stimuli that are received determine their response during development, myelination and injuries. To this end, the mechanobiological response of SCs is being actively researched, as it can determine the suitability of fabricated scaffolds for tissue engineering and regenerative medicine applications. There is growing evidence that SCs are sensitive to changes in the mechanical properties of the surrounding environment (such as the type of material, its elasticity and stiffness), different topographical features provided by the environment, as well as shear stress. In this review, we explore how different mechanical stimuli affect SC behaviour and highlight the importance of exploring many different avenues when designing scaffolds for the repair of PNS injuries.

A biomaterials approach to Schwann cell development in neural tissue engineering.

Schwann cells, in addition to forming myelin sheaths, have pivotal roles in regeneration of injured axons in the peripheral nervous system such as producing a natural permissive conduit between distal and proximal stumps and secreting nerve growth factors. Due to the atrophy and senescence of Schwann cells in long nerve gap, and the need to ensure the presence of nerve growth factors and basal lamina tubes for axon regeneration in a critical time, injection of Schwann cells with the aid of an engineered conduit seems to be an effective approach to induce axon regrowth. Stem cells with high differentiation and proliferation capability can provide an adequate number of Schwann cells in healthy state for regeneration purposes. Guidance of stem cells differentiation into desired lineages, control of implanted Schwann cells fate, maintenance of nerve growth factors expression, and guidance of axon regrowth are possible with the aid of biomaterials with appropriate chemical, physical, and mechanical properties. Biomaterials' surface chemistry and biomolecules interacting with cells' receptors initiate specific intracellular signaling cascades and direct cells fate. In addition, biomaterials' surface topography in association with cells contact area, focal adhesion, and cytoskeletal remodeling by mechanotransduction process influences cells behavior and induces specific differentiation. The main objective of this review is to investigate the chemical, topographical, and mechanical properties of biomaterials which influence the fate of Schwann cells and the nerve regeneration process.

Environmental cues determine the fate of astrocytes after spinal cord injury.

Reactive astrogliosis occurs after central nervous system (CNS) injuries whereby resident astrocytes form rapid responses along a graded continuum. Following CNS lesions, naïve astrocytes are converted into reactive astrocytes and eventually into scar-forming astrocytes that block axon regeneration and neural repair. It has been known for decades that scarring development and its related extracellular matrix molecules interfere with regeneration of injured axons after CNS injury, but the cellular and molecular mechanisms for controlling astrocytic scar formation and maintenance are not well known. Recent use of various genetic tools has made tremendous progress in better understanding genesis of reactive astrogliosis. Especially, the latest experiments demonstrate environment-dependent plasticity of reactive astrogliosis because reactive astrocytes isolated from injured spinal cord form scarring astrocytes when transplanted into injured spinal cord, but revert in retrograde to naive astrocytes when transplanted into naive spinal cord. The interactions between upregulated type I collagen and its receptor integrin β1 and the N-cadherin-mediated cell adhesion appear to play major roles for local astrogliosis around the lesion. This review centers on the environment-dependent plasticity of reactive astrogliosis after spinal cord injury and its potential as a therapeutic target.

Neural stem cell transplantation combined with erythropoietin for the treatment of spinal cord injury in rats

Spinal cord injury (SCI) comprises nerve and motor function disorders that may be caused by a variety of damaging factors and is challenging to treat. The aim of the present study was to investigate the regenerative effects of neural stem cell (NSC) transplantation combined with intraperitoneal injection of erythropoietin (EPO) on cross-sectional SCI in rats. A model of SCI was induced in 40 adult Wistar rats via the complete transection of the 10th thoracic vertebra (T10). The rats were allocated at random into 4 groups: Control, NSC, EPO and NSC + EPO groups (n=10 per group). Morphological alterations associated with axonal regeneration were detected using neurofilament (NF)-200 immunohistochemistry and immunofluorescence staining after 8 weeks. Basso, Beattie and Bresnahan (BBB) scoring was used to evaluate the recovery of hindlimb function. A total of 5 rats died following surgery, including 2 control rats and 1 rat each in the EPO, NSC and NSC + EPO groups. NSCs labeled with bromodeoxyuridine were observed to have survived and migrated in the spinal cord tissue after 8 weeks. Significant histomorphological differences were observed in the NSC and NSC + EPO groups compared with the EPO and control groups. Furthermore, the rats of the NSC + EPO group exhibited significantly enhanced axonal regeneration in the SCI area compared with the NSC group rats. The rats of the NSC and NSC + EPO groups exhibited significantly improved BBB scores compared with the EPO and control group rats at 7 days after treatment (P<0.05). In addition, the BBB scores of the NSC + EPO group were significantly improved compared with those of the three other groups at 7 days after surgery (P<0.05). Therefore, the results of the present study suggest that NSC transplantation combined with intraperitoneal injection of EPO may benefit the survival and regeneration of injured axons, and accelerate the repair of injured spinal cord tissue, thus facilitating the functional recovery of hindlimb locomotor function in rats.

Nogo-p4 Suppresses TrkA Signaling Induced by Low Concentrations of Nerve Growth Factor Through NgR1 in Differentiated PC12 Cells

Background: Regeneration of injured axons in adult mammalian central nervous system (CNS) is not spontaneous. Nogo is a major inhibitory molecule contributing to axon regeneration failure. The molecular mechanisms of Nogo inhibition of axon regeneration are not completely understood. To further investigate the underlying mechanisms, we studied the effects of Nogo-p4, a 25-amino acid core inhibitory fragment of Nogo, on nerve growth factor (NGF)-induced TrkA signaling. Methods: NGF-differentiated PC12 cells were used as cell models. The effects of Nogo-p4 on two key components of TrkA signaling, phosphorylated Erk1/2 and Akt, were analyzed by western blot. Co-immunoprecipitation experiments were performed to detect the formation of NgR1/p75 complexes. Neurite growth was quantified by measuring the neurite length. Results: Nogo-p4 did not significantly affect TrkA signaling induced by 100 ng/ml NGF, but signaling was suppressed when an NGF concentration of 5 ng/ml was used. Further investigation demonstrated that Nogo-p4 affected TrkA signaling in an NGF concentration-dependent manner. Nogo-p4 suppression of TrkA signaling was strong at low (1 and 5 ng/ml), moderate at intermediate (25 ng/ml), but absent at high (50 and 100 ng/ml) NGF concentrations. NEP1-40 attenuated, and NgR1 overexpression enhanced, Nogo-p4 suppression of TrkA signaling induced by low concentrations of NGF. High but not low concentrations of NGF reduced the formation of NgR1/p75 complexes triggered by Nogo-p4. Nogo-p4 strongly inhibited neurite growth induced by low rather than high concentrations of NGF. Conclusion: Nogo-p4 binding with NgR1 suppresses TrkA signaling induced by low concentrations of NGF in differentiated PC12 cells. Suppression of NGF-induced TrkA signaling may be another mechanism by which Nogo inhibits neurite growth.

Epigenomic reprogramming in peripheral nerve injury.

Epigenomic reprogramming in peripheral nerve injury.

Guiding the migration of grafted cells to promote axon regeneration.

A promising therapeutic strategy to promote the regeneration of injured axons in the adult central nervous system (CNS) is the transplantation of cells or tissues that can modify the local host environment and support the growth of regenerating axons. Growth-supportive cells that have been successfully used in experimental transplantation therapy of spinal cord injury (SCI) include Schwann cells, mesenchymal stromal cells, olfactory ensheathing cells, genetically modified fibroblasts, and neural stem/progenitor cells (Huang et al., 2010). Cells derived from the embryonic spinal cord and peripheral nerve grafts have been shown to promote the regeneration of injured axons, due largely to the presence of growth-supportive cells such as glial progenitors and Schwann cells, respectively (Cote et al., 2011; Haas and Fischer, 2013). These transplants generate a permissive environment for axon growth by secreting growth factors and forming an adhesive extracellular matrix to overcome the inhibitory environment of the injured tissue. However, the value of these transplants to promote axon regeneration is limited by the fact that most regenerating axons are trapped inside the permissive environment generated by the transplants, failing to grow out of the graft (Figure ​Figure1A,1A, ​,BB) (Haas and Fischer, 2013). While this strategy can be effective for building functional relays via graft-derived neurons (Haas and Fischer, 2014), this approach can not be generalized to other cell types. Therefore, a remaining challenge for therapeutic cell transplantation in CNS injury, in the context of long distance regeneration and connectivity, is to develop strategies to promote axonal growth beyond the graft into putative target areas to form functional synaptic connections. Currently the nature of the “graft trap” of regenerating axons is not fully understood. One possibility is that the regenerating axons stay inside the graft, which expresses much higher levels of attractive guidance factors, i.e., neurotrophic factors, and much lower levels of inhibitory/repulsive factors, i.e., chondroitin sulfate proteoglycan (CSPG), compared to the adjacent host tissue. Another possibility is that although adult CNS axons maintain their growth potential and can regenerate in an optimized environment, their intrinsic growth capability is much lower than axons of embryonic neurons, and thus not suitable for long-distance regeneration. Targeting these mechanisms, several strategies have recently been applied to overcome the “graft trap” in transplantation-based therapy of SCI. One strategy is to further modify host spinal tissue, making the host tissue less inhibitory and thus allowing some of the regenerating axons inside the graft to exit into the host tissue. As an example, Tom et al. (2009) showed that in an experimental model of grafting a peripheral nerve bridge at the site of the injured spinal cord, application of chondroitinase (Chase) at the distal graft/host interface to reduce CSPG-mediated inhibition promoted modest improvement in host-entry of regenerating axons, which would otherwise stop at the distal graft/host junction. Another strategy to promote axonal growth beyond the graft focuses on genetic modification of injured neurons to enhance their intrinsic growth potential using regeneration associated genes (Ma and Willis, 2015). For example, overexpressing the constitutively active form of the Rheb GTPase (downstream of the mTOR pathway) has been shown to enhance the intrinsic growth potential of adult neurons (Wu et al., 2015).

Regeneration of sensory but not motor axons following visceral nerve injury

Following peripheral nerve injury, restoration of function may occur via the regeneration of injured axons or compensatory sprouting of spared axons. Injury to visceral nerves that control urogenital organs is a common consequence of pelvic surgery, however their capacity to reinnervate organs is poorly understood. To determine if and how sensory and motor connections to the bladder are re-established, a novel surgical model of visceral nerve injury was performed unilaterally in adult male Wistar rats. Bladder-projecting motor and sensory neurons in pelvic ganglia and lumbosacral dorsal root ganglia, respectively, were identified and characterised by retrograde tracing and immunofluorescence. Application of tracers ipsi- and contralateral to injury distinguished the projection pathways of new connections in the bladder. In naive animals, the majority of sensory and motor neurons project ipsilaterally to the bladder, while ~20 % project contralaterally and ~5 % bilaterally. Injured axons of motor neurons were unable to regenerate by 4weeks after transection. In contrast, by this time many injured sensory neurons regrew axons to reform a substantial plexus within the detrusor and suburothelial tissues. These regeneration responses were also indicated by upregulation of activating transcription factor-3 (ATF-3), which was sustained in motor neurons but transient in sensory bladder-projecting neurons. Axotomy had little or no effect on the survival of bladder-projecting sensory and motor neurons. We also found evidence that uninjured motor and sensory neurons develop additional projections to the denervated bladder tissue and return connectivity, likely by undergoing compensatory growth. In conclusion, our results show that visceral sensory and motor neurons have a different capacity to regenerate axons following axotomy, however in both components of the circuit uninjured bladder neurons spontaneously grow new axon collaterals to replace the lost terminal field within the organ. For a full functional recovery, understanding the environmental and cellular mechanisms that reduce the ability of pelvic ganglion cells to undergo axonal regeneration is needed.

Assessment of sensory thresholds and nociceptive fiber growth after sciatic nerve injury reveals the differential contribution of collateral reinnervation and nerve regeneration to neuropathic pain

Following traumatic peripheral nerve injury reinnervation of denervated targets may be achieved by regeneration of injured axons and by collateral sprouting of neighbor undamaged axons. Experimental models commonly use sciatic nerve injuries to assess nerve regeneration and neuropathic pain, but behavioral tests for evaluating sensory recovery often disregard the pattern of hindpaw innervation. This may lead to confounding attribution of recovery of sensory responses to improvement in sciatic nerve regeneration instead of collateral reinnervation by the undamaged saphenous nerve. We used a standardized methodology to assess the separate contribution of collateral and regenerative skin reinnervation on sensory responses. Section and suture of the sciatic nerve induced loss of sensibility in the lateral and central areas of the injured paw, but nociceptive responses rapidly recovered by expansion of the intact saphenous innervation territory. We used electronic Von Frey and Plantar test devices to measure mechanical and thermal withdrawal thresholds in specific sites of the injured paw: lateral site innervated by the sciatic nerve, medial site that remained innervated by the saphenous nerve, and central site originally innervated by the sciatic nerve but affected by saphenous sprouting. After sciatic section, signs of early hyperalgesia developed in medial and central paw areas due to saphenous sprouting and expansion. The regenerating sciatic nerve fibers reached the paw at 3–4weeks and a late mechanical hyperalgesia was observed at the lateral site. Immunohistochemical staining of sensory fibers innervating the medial and lateral areas revealed a different pattern of skin reinnervation. Hypersensitivity in the intact saphenous nerve area was paralleled by early fiber sprout growth in the subepidermal plexus, but not entering the epidermis. On the other side, late sciatic hyperalgesia was accompanied by gradual skin reinnervation after 4weeks.The standardization of algesimetry testing in sciatic nerve injury models, as proposed in this study, provides a suitable model for studying in parallel neuropathic pain and sensory nerve regeneration processes. Our results also indicate that collateral sprouting and axonal regeneration contribute differently in the initiation and maintenance of neuropathic pain.

Quantitative assessment of neurite outgrowth in mouse retinal explants.

Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system (CNS) remains elusive. The discovery of novel neuro-stimulatory agents that promote regeneration is hampered by a gap between high content analysis platforms using neuronal cells and time-consuming preclinical animal models. In this regard, tissue explant cultures, which are easily manageable and more closely resemble the in vivo situation, form an ideal model system to study the effect of compounds on the neuroglial network. Retinal explants have proven to be a useful tool to investigate the effect of molecules on neuronal survival and regeneration. In this chapter, we report a detailed description of how to isolate and culture retinal explants and how to immunolabel the outgrowing neurites. Furthermore, we describe different analysis tools, both manual and automated, to quantify neurite outgrowth from retinal explants.

Repairing the injured spinal cord: sprouting versus regeneration. Is this a realistic match?

Repairing the injured spinal cord: sprouting versus regeneration. Is this a realistic match?

Automated Analysis of Neurite Outgrowth in Mouse Retinal Explants

Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system remains elusive. In the quest for neurostimulatory agents that promote regeneration, well-defined models and analysis methods are required. Tissue explant cultures closely resemble the in vivo situation, making them ideal to study the effect of compounds on the neuro-glial network. This study reports the optimization of an explant culture technique using retinas of neonatal mice and the development of an analysis script that allows for rapid and automated analysis of neurite outgrowth from these explants. The key features of this script (i.e., local thresholding and form selection) allow for swift and unbiased detection of neurite outgrowth. The novel analysis method is compared with two commonly used manual methods and successfully validated by performing dose-response studies with molecules known to either inhibit (anti–β1-integrin antibody) or stimulate (brain-derived neurotrophic factor and ciliary neurotrophic factor) neurite outgrowth from retinal explants. Finally, the new analysis script is used to study whether retinal explant origin has any effect on neurite outgrowth.

Uncovering New Pharmacological Targets to Treat Neuropathic Pain by Understanding How the Organism Reacts to Nerve Injury

The neuropathic pain syndrome is complex. Current drugs to treat neuropathic pain, including anticonvulsivants and antidepressants, fail in up to 40-50% of the patients, while in the rest of them total alleviation is not normally achieved. Increased research advances in the neurobiology of neuropathic pain have not translated in more successful pharmacological treatments by the moment, but recent progress in the experimental methods available for this purpose could result in significant advances in the short term. One rational possibility for the pharmaceutical development of new drugs, including target identification, drug design and evaluation studies, could be to focus on mimicking what organism does to limit nerve damage or to enhance the regeneration of injured axons. Following this strategy, neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been postulated as potential pharmacological targets to treat neuropathic pain. In addition, during the last few years, strong scientific evidences point to novel neurotrophic factors, such as pleiotrophin (PTN), as important factors to limit neuropathic pain development because of their remodeling and angiogenic actions in the injured area. This review focuses on recent research advances identifying new pharmacological targets in the treatment of the cause, not only the symptoms, of neuropathic pain.

Assessing Spinal Axon Regeneration and Sprouting in Nogo-, MAG-, and OMgp-Deficient Mice

A central hypothesis for the limited capacity for adult central nervous system (CNS) axons to regenerate is the presence of myelin-derived axon growth inhibitors, the role of which, however, remains poorly understood. We have conducted a comprehensive genetic analysis of the three major myelin inhibitors, Nogo, MAG, and OMgp, in injury-induced axonal growth, including compensatory sprouting of uninjured axons and regeneration of injured axons. While deleting any one inhibitor in mice enhanced sprouting of corticospinal or raphespinal serotonergic axons, there was neither associated behavioral improvement nor a synergistic effect of deleting all three inhibitors. Furthermore, triple-mutant mice failed to exhibit enhanced regeneration of either axonal tract after spinal cord injury. Our data indicate that while Nogo, MAG, and OMgp may modulate axon sprouting, they do not play a central role in CNS axon regeneration failure.

Disinhibition of neurite growth to repair the injured adult CNS: focusing on Nogo.

Investigations into mechanisms that restrict the recovery of functions after an injury to the brain or the spinal cord have led to the discovery of specific neurite growth inhibitory factors in the adult central nervous system (CNS) of mammals. Blocking their growth-suppressive function resulted in disinhibition of axonal growth, i.e. growth of cultured neurons on inhibitory CNS tissue in vitro and regeneration of injured axons in vivo. The enhanced regenerative and compensatory fibre growth was often accompanied by a substantial improvement in the functional recovery after CNS injury. The first clinical studies to assess the therapeutic potential of compounds that neutralize growth inhibitors or interfere with their downstream signalling are currently in progress. This review discusses recent advances in the understanding of how the 'founder molecule' Nogo-A and other glial-derived growth inhibitors restrict the regeneration and repair of disrupted neuronal circuits, thus limiting the functional recovery after CNS injuries.

Myelin‐associated glycoprotein reduces axonal branching and enhances functional recovery after sciatic nerve transection in rats

The mature peripheral nervous system (PNS) generally shows better regeneration of injured axons as opposed to the central nervous system (CNS). However, complete functional recovery is rarely achieved even in the PNS although morphologically good axonal regeneration often occurs. This mainly results from aberrant reinnervation due to extensive branching of cut axons with consequent failure of synchronized movements of the muscles. Myelin-associated glycoprotein (MAG), a well-characterized molecule existing both in the CNS and PNS myelin, is considered to be a potent inhibitor of axonal regeneration especially in the CNS. In the present study, we investigated whether MAG has any effects not only on axonal elongation, but also on axonal branching. We show herein that MAG minimized branching of the peripheral axons both in vitro and in vivo via activation of RhoA. Furthermore, after sciatic nerve transection in rats, focal and temporary application of MAG to the lesion dramatically enhanced the functional recovery. Using double retrograde labeling and preoperative/postoperative labeling of spinal neurons, reduced hyperinnervation and improved accuracy of target reinnervation was confirmed, respectively. In conclusion, as MAG significantly improves the quality of axonal regeneration, it can be used as a new therapeutic approach for peripheral nerve repair with possible focal and temporary application.

Natural product promotes repair of injured spinal cord

The regenerative capacity of axons in the mammalian CNS is poor relative to other tissues in the body such as the skin or bone. In contrast, regeneration of injured axons in the peripheral nervous system is generally more robust, which has suggested that locally produced CNS-derived growth inhibitors prevent axonal regeneration. Semaphorin3A (Sema3A) is a key inhibitor of axonal regeneration and a small-molecule inhibitor of Sema3A (SM-216289, isolated as a natural product from fungal fermentation) has been shown to promote CNS repair in adult rats subjected to spinal cord transection. Treatment with SM-216289 caused increased regeneration of injured axons, Schwann cell-mediated myelination, enhanced angiogenesis and decreased apoptotic cell death. This study validates both Sema3A as a therapeutic target and suggests that optimisation of compounds based on this pharmacophore may have use in the treatment of spinal cord injury in man.

BDNF promotes connections of corticospinal neurons onto spared descending interneurons in spinal cord injured rats

Although regeneration of injured axons is inhibited within the adult CNS, moderate recovery can be found in patients and animals with incomplete spinal cord injury (SCI). This can be partly attributed to sprouting of spared and injured axons, rostral and caudal to the lesion, respectively. Recently, it has been reported that following a thoracic SCI such sprouting can result in indirect reconnections of the lesioned axons to caudal targets via propriospinal interneurons (PrI). Here, we attempted to further promote this spontaneous repair mechanism by applying the neurotrophic factor BDNF (brain-derived neurotrophic factor), in the vicinity of the cell bodies of lesioned corticospinal neurons or NT-3, intrathecally to the cervical spinal cord. We performed a dorsal over-hemisection at the thoracic spinal cord sparing only the left ventrolateral quadrant. This type of lesion did not promote sprouting of injured corticospinal axons or re-routing via commissural PrI. Also, in rats that received NT-3 at the cervical enlargement, no increase in sprouting was found. However, animals receiving BDNF at the cell bodies of lesioned corticospinal neurons showed a significant increase in collateral sprouting and in the number of contacts with PrI. This was not observed when BDNF was administered to unlesioned animals. Although no statistical difference in the horizontal ladder walking was found between the groups, the increase in collateral sprouting and in the number of contacts correlated with the functional recovery. Hence, cell body treatment can promote plasticity of the injured CNS and may be a valuable treatment approach in conjunction with local regeneration promoting strategies.

Targeting Myelin to Optimize Plasticity of Spared Spinal Axons

Functional re-innervation of target neurons following neurological damage such as spinal cord injury is an essential requirement of potential therapies. There are at least two avenues by which this can be achieved: (a) through the regeneration of injured axons and (b) through promoting plasticity of those spared by the initial insult. There are several reasons why the latter approach may be more feasible, not the least of which are the inhibitory character of the glial scar, the often long distances over which injured axons must regrow, and the fact that spared axons are often already in the vicinity of denervated targets. The challenge is to unveil the well-recognized intrinsic plasticity of spared axons in a way that avoids complications, such as pain or autonomic dysfunction. One approach that we as well as others have taken is to target growth-suppressing signaling pathways initiated in spared axons by myelin-derived proteins. This article reviews models used for the study of spinal axon plasticity and describes the anatomical and behavioral effects of interfering with myelinderived proteins, their receptors, and components of their intracellular signaling cascades.