Regeneration Of Injured Neurons Research Articles

Skin Reinnervation by Collateral Sprouting Following Spared Nerve Injury in Mice.

Following peripheral nerve injury, denervated tissues can be reinnervated via regeneration of injured neurons or collateral sprouting of neighboring uninjured afferents into denervated territory. While there has been substantial focus on mechanisms underlying regeneration, collateral sprouting has received less attention. Here, we used immunohistochemistry and genetic neuronal labeling to define the subtype specificity of sprouting-mediated reinnervation of plantar hindpaw skin in the mouse spared nerve injury (SNI) model, in which productive regeneration cannot occur. Following initial loss of cutaneous afferents in the tibial nerve territory, we observed progressive centripetal reinnervation by multiple subtypes of neighboring uninjured fibers into denervated glabrous and hairy plantar skin of male mice. In addition to dermal reinnervation, CGRP-expressing peptidergic fibers slowly but continuously repopulated denervated epidermis, Interestingly, GFRα2-expressing nonpeptidergic fibers exhibited a transient burst of epidermal reinnervation, followed by a trend towards regression. Presumptive sympathetic nerve fibers also sprouted into denervated territory, as did a population of myelinated TrkC lineage fibers, though the latter did so inefficiently. Conversely, rapidly adapting Aβ fiber and C fiber low threshold mechanoreceptor (LTMR) subtypes failed to exhibit convincing sprouting up to 8 weeks after nerve injury in males or females. Optogenetics and behavioral assays in male mice further demonstrated the functionality of collaterally sprouted fibers in hairy plantar skin with restoration of punctate mechanosensation without hypersensitivity. Our findings advance understanding of differential collateral sprouting among sensory neuron subpopulations and may guide strategies to promote the progression of sensory recovery or limit maladaptive sensory phenomena after peripheral nerve injury.

Chondroitin Sulfate Glycosaminoglycans Regulate Distinct Cell Surface Receptor-Mediated Neuronal Functions

Chondroitin sulfate (CS) chains, a class of sulfated glycosaminoglycan (GAG) polysaccharides, are major extra/pericellular components that function as microenvironmental cues during neuronal development and the regeneration of injured neurons. Interestingly, CS chains exert both facilitatory and inhibitory effects on neurite outgrowth. Such bidirectional CS chain effects are ascribable to their own structural divergence that could be generated via distinct enzymatic modifications. CS chains have long been considered as passive structural scaffolds that often behave as co-receptors and/or reservoirs for various humoral factors. However, the recent identification of cell surface receptor molecules with binding preferences to distinct CS structures has provided new insight into a novel CS chain effector mechanism functioning as dynamic extra/pericellular signaling ligands that could directly control the corresponding receptor-mediated signaling pathways. This new concept might also support the functional complexity of CS chains in neurobiological events.

Genome-wide analyses of psychological resilience in U.S. Army soldiers.

Though a growing body of preclinical and translational research is illuminating a biological basis for resilience to stress, little is known about the genetic basis of psychological resilience in humans. We conducted genome-wide association studies (GWASs) of self-assessed (by questionnaire) and outcome-based (incident mental disorders from predeployment to postdeployment) resilience among European (EUR) ancestry soldiers in the Army study to assess risk and resilience in servicemembers. Self-assessed resilience (N = 11,492) was found to have significant common-variant heritability (h2 = 0.162, se = 0.050, p = 5.37 × 10-4 ), and to be significantly negatively genetically correlated with neuroticism (rg = -0.388, p = .0092). GWAS results from the EUR soldiers revealed a genome-wide significant locus on an intergenic region on Chr 4 upstream from doublecortin-like kinase 2 (DCLK2) (four single nucleotide polymorphisms (SNPs) in LD; top SNP: rs4260523 [p = 5.65 × 10-9 ] is an eQTL in frontal cortex), a member of the doublecortin family of kinases that promote survival and regeneration of injured neurons. A second gene, kelch-like family member 36 (KLHL36) was detected at gene-wise genome-wide significance [p = 1.89 × 10-6 ]. A polygenic risk score derived from the self-assessed resilience GWAS was not significantly associated with outcome-based resilience. In very preliminary results, genome-wide significant association with outcome-based resilience was found for one locus (top SNP: rs12580015 [p = 2.37 × 10-8 ]) on Chr 12 downstream from solute carrier family 15 member 5 (SLC15A5) in subjects (N = 581) exposed to the highest level of deployment stress. The further study of genetic determinants of resilience has the potential to illuminate the molecular bases of stress-related psychopathology and point to new avenues for therapeutic intervention.

Perspectives of TRPV1 Function on the Neurogenesis and Neural Plasticity.

The development of new strategies to renew and repair neuronal networks using neural plasticity induced by stem cell graft could enable new therapies to cure diseases that were considered lethal until now. In adequate microenvironment a neuronal progenitor must receive molecular signal of a specific cellular context to determine fate, differentiation, and location. TRPV1, a nonselective calcium channel, is expressed in neurogenic regions of the brain like the subgranular zone of the hippocampal dentate gyrus and the telencephalic subventricular zone, being valuable for neural differentiation and neural plasticity. Current data show that TRPV1 is involved in several neuronal functions as cytoskeleton dynamics, cell migration, survival, and regeneration of injured neurons, incorporating several stimuli in neurogenesis and network integration. The function of TRPV1 in the brain is under intensive investigation, due to multiple places where it has been detected and its sensitivity for different chemical and physical agonists, and a new role of TRPV1 in brain function is now emerging as a molecular tool for survival and control of neural stem cells.

Doublecortin-Like Kinases Promote Neuronal Survival and Induce Growth Cone Reformation via Distinct Mechanisms

After axotomy, neuronal survival and growth cone re-formation are required for axon regeneration. We discovered that doublecortin-like kinases (DCLKs), members of the doublecortin (DCX) family expressed in adult retinal ganglion cells (RGCs), play critical roles in both processes, through distinct mechanisms. Overexpression of DCLK2 accelerated growth cone re-formation in vitro and enhanced the initiation and elongation of axon re-growth after optic nerve injury. These effects depended on both the microtubule (MT)-binding domain and the serine-proline-rich (S/P-rich) region of DCXs in-cis in the same molecules. While the MT-binding domain is known to stabilize MT structures, we show that the S/P-rich region prevents F-actin destabilization in injured axon stumps. Additionally, while DCXs synergize with mTOR to stimulate axon regeneration, alone they can promote neuronal survival possibly by regulating the retrograde propagation of injury signals. Multifunctional DCXs thus represent potential targets for promoting both survival and regeneration of injured neurons.

Regeneration of optic nerve fibers of adult mammals

The pathway from the retina to the brain in mammals provides a well-defined model system for investigation of not only surviving axotomy but also axonal regeneration of injured neurons. Here I introduce our recent works on axonal regeneration in the optic nerve (OpN) of adult cats. Fibers of retinal ganglion cells (RGCs) extend beyond the crush site of OpN with injections of a macrophage stimulator (oxidized galectin-1) or a Rho kinase (ROCK) inhibitor (Y-39983 or Y-27632) while axonal extension is blocked with injection of saline. Elongation of crushed optic fibers, however, is slowed after 2 weeks. Transplantation of peripheral nerve makes RGCs regenerate their transected axons into a graft but regenerated fibers extend only a few mm in the brain. Effectiveness of combination of the drugs and treatments has to be verified in future.

Axon regeneration requires a conserved MAP kinase pathway.

Regeneration of injured neurons can restore function, but most neurons regenerate poorly or not at all. The failure to regenerate in some cases is due to a lack of activation of cell-intrinsic regeneration pathways. These pathways might be targeted for the development of therapies that can restore neuron function after injury or disease. Here, we show that the DLK-1 mitogen-activated protein (MAP) kinase pathway is essential for regeneration in Caenorhabditis elegans motor neurons. Loss of this pathway eliminates regeneration, whereas activating it improves regeneration. Further, these proteins also regulate the later step of growth cone migration. We conclude that after axon injury, activation of this MAP kinase cascade is required to switch the mature neuron from an aplastic state to a state capable of growth.

Umbilical cord blood mesenchymal stromal cells are neuroprotective and promote regeneration in a rat optic tract model

Exploitation of the ability of stem cells to protect damaged neuronal tissue may be a more viable strategy than cell replacement for repair of the central nervous system (CNS). In this study we assessed the capacity of human umbilical cord blood (hUCB)-derived mesenchymal stromal cells (MSCs) to protect and promote regeneration of axotomised neurons within the rat optic system. The optic tract of neonatal rats was transected at the level of the lateral geniculate nucleus, and MSCs were introduced into the lesion site. MSCs survived well up to 2 weeks after grafting, and did not migrate significantly or differentiate. In the presence of MSC grafts, host axonal processes were found to be present in the lesion site, and there was stimulation of an endogenous neural precursor population. Four weeks after grafting, retrograde tracer experiments demonstrated that grafted MSCs, as well as cells of a human fibroblast line, exerted a neuroprotective effect, rescuing a significant percentage of axotomised retinal ganglion cells (RGCs). Further experiments with retrograde and anterograde tracers strongly indicated that MSCs could also promote re-growth of axotomised RGCs to their target, the superior colliculus (SC). Further analysis showed that hUCB-derived MSCs secreted several immunomodulatory and neurotrophic factors in vitro, including TGFβ1, CNTF, NT-3 and BDNF, which are likely to play a role in neuroprotection. Our data indicate that hUCB-derived MSCs may be an easily accessible, widely available source of cells that can contribute towards neural repair through rescue and regeneration of injured neurons.

Identification of Peripherin as a Akt Substrate in Neurons

Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study, we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt. PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser(66) in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK 293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration.

Research on characteristics of neuron potassium channel under low level laser irradiation

Acutely isolated rat hippocampal CA3 pyramidal neurons were irradiated with a semiconductor laser of wavelength 670 nm and power 5 mW. The properties of transient outward potassium (K+) channel were investigated using the whole-cell patch clamp technique. The experiment revealed that low level laser reduced the amplitudes of IA in a voltage-dependent and reversible manner. The steady-state activation and inactivation processes of IA were significantly affected by laser irradiation. And the steady-state activation curve shifted towards positive potential direction, altering the slope factor of curve and increasing the half-activation voltage. However, the steady-state inactivation curve shifted towards negative potential direction, reducing the half-inactivation voltage without changing the slope factor. The results show that low level laser irradiation could change the characteristics of transient outward K+ channel. Accordingly, the firing of action potential and the physiological function of neurons were adjusted as a result of low level laser irradiation, which might contribute to the recovery and regeneration of injured neurons.

Growth and guidance cues for regenerating axons: where have they gone?

Both attractive and repellent cues are required to guide developing axons to their targets in the central nervous system. Critical guidance molecules in the developing brain include the semaphorins, netrins, slits, and ephrins. Current research indicates that many of these molecules and their receptors are expressed in the adult central nervous system (CNS), and that injury can alter the levels of these ligands/receptors. Recent studies have begun the process of elucidating the functions of these receptors in adult mammals, and the effects that they have on the regeneration of adult neurons. This review addresses our current knowledge with respect to the response of adult CNS neurons to axonal injury, interventions for enhancing the survival and regeneration of injured neurons, and the expression of developmental axon guidance cues in the injured mature CNS, with specific focus on the retino-tectal projection.

Viral vector-mediated gene transfer of neurotrophins to promote regeneration of the injured spinal cord

Injuries to the adult mammalian spinal cord often lead to severe damage to both ascending (sensory) pathways and descending (motor) nerve pathways without the perspective of complete functional recovery. Future spinal cord repair strategies should comprise a multi-factorial approach addressing several issues, including optimalization of survival and function of spared central nervous system neurons in partial lesions and the modulation of trophic and inhibitory influences to promote and guide axonal regrowth. Neurotrophins have emerged as promising molecules to augment neuroprotection and neuronal regeneration. Although intracerebroventricular, intrathecal and local protein delivery of neurotrophins to the injured spinal cord has resulted in enhanced survival and regeneration of injured neurons, there are a number of drawbacks to these methods. Viral vector-mediated transfer of neurotrophin genes to the injured spinal cord is emerging as a novel and effective strategy to express neurotrophins in the injured nervous system. Ex vivo transfer of neurotrophic factor genes is explored as a way to bridge lesions cavities for axonal regeneration. Several viral vector systems, based on herpes simplex virus, adenovirus, adeno-associated virus, lentivirus, and moloney leukaemia virus, have been employed. The genetic modification of fibroblasts, Schwann cells, olfactory ensheathing glia cells, and stem cells, prior to implantation to the injured spinal cord has resulted in improved cellular nerve guides. So far, neurotrophic factor gene transfer to the injured spinal cord has led to results comparable to those obtained with direct protein delivery, but has a number of advantages. The steady advances that have been made in combining new viral vector systems with a range of promising cellular platforms for ex vivo gene transfer (e.g., primary embryonic neurons, Schwann cells, olfactory ensheating glia cells and neural stem cells) holds promising perspectives for the development of new neurotrophic factor-based therapies to repair the injured nervous system.

Neurotrophic factors in central nervous system trauma.

Although regeneration of injured neurons does not occur after trauma in the central nervous system (CNS), there is often significant recovery of functional capacity with time. Little is currently known about the molecular basis for such recovery, but the increased trophic activity in injured CNS tissue and the known properties of neurotrophic factors in neuronal growth and maintenance suggest that these polypeptides are probably involved in recovery of function. Members of the neurotrophin family, including nerve growth factor (NGF), brain-derived neurotrophic factors (BDNF), and neurotrophin 3 (NT-3), are capable of supporting survival of injured CNS neurons both in vitro and in vivo. They also stimulate neurite outgrowth, needed for reorganization of the injured CNS, and the expression of key enzymes for neurotransmitter synthesis that may need to be upregulated to compensate for reduced innervation. The effects of the neurotrophins are mediated through specific high affinity trk receptors (trk A, B, C) as well as a common low affinity receptor designated p75NGFR. Another class of neurotrophic polypeptides also provides candidate recovery-promoting molecules, the heparin-binding growth factors' acidic and basic fibroblast growth factor (aFGF, bFGF). FGFs not only sustain survival of injured neurons but also stimulate revascularization and certain glial responses to injury. Both the neurotrophins and the FGFs, as well as their respective receptors, have been shown to be upregulated after experimental CNS injury. Further, administration of neurotrophins or FGF has been shown to reduce the effects of experimental injury induced by axotomy, excitotoxins, and certain other neurotoxins. The cellular basis for the potential therapeutic use of neurotrophic molecules is discussed as well as new strategies to increase neurotrophic activity after CNS trauma based on the recently obtained information on pharmacological and molecular control of the expression of these genes.

Axonal regeneration after peripheral nerve grafting and fibrin–fibronectin-containing matrix implantation on the injured septohippocampal pathway of the adult rat: a light and electron microscopic study

The damaged septohippocampal pathway was utilized to study the axonal regeneration of injured neurons. Semipermeable tubes, 2-mm long, were placed in the axis of the transected septohippocampal pathway of adult rats. In a first series of experiments, empty tubes were implanted. Even six weeks after the operation, no regenerated axons were observed in the conduit. In a second series of experiments, in order to validate our approach, segments of pre-degenerated sciatic nerves were introduced into the tubes. Under these experimental conditions, acetylcholinesterase (AChE)-containing regenerated axonal processes were detected in the grafted sciatic nerves. Glial fibrillary acidic protein (GFAP)-immunodetection showed that astroglial cells and astrocyte processes were able to progress on and into the peripheral grafts. At the electron microscopic level, axons were observed in close contact with Schwann cells which myelinated some of them. In some other cases, unmyelinated axons were also present at the surface of reactive astroglial cells filled by numerous intermediate filaments. These central glial cells had migrated among the sciatic nerve collagen fibers. No axon was detected without glial cell contact. In a third series of experiments, we implanted semipermeable tubes previously filled with a fibrin-fibronectin-containing matrix provided by peripheral regeneration chambers. One week after the implantation of the tubes containing this peripheral substrate, different cell types were observed migrating into the conduit and replacing the fibrin-fibronectin-containing matrix. Among these cells astrocytes were present as revealed by GFAP-immunocytochemistry and electron microscopic examinations. During the following weeks, axons were detected in contact with the reactive astroglial cells. AChE-histochemistry showed that axons were able to cross the two millimeter distance separating the septal part and the hippocampal part of the lesion site. GABA (γ-aminobutyric acid)-ergic fibers were also detected in the regenerated structure. These experiments show that cellular or acellular substrates provided by the PNS can promote the regeneration of CNS GABAergic and cholinergic neurons. Our observations suggest that astrocytes can take an important part, after their migration or after extending processes, in the axonal regeneration in the adult CNS of the rat, possibly in furnishing a cellular terrain for the progression of growth cones over a distance of two millimeters and in maintaining regenerated axons at least until the sixth week after the operation.