RESUMO
BACKGROUND: Salidroside is the major bioactive and pharmacological active substance in Rhodiola rosea L. It has been reported to have neuroprotective effects on cerebral ischemia/reperfusion (I/R). However, whether salidroside can enhance neural regeneration after cerebral I/R is still unknown. This study investigated the effects of salidroside on the endogenous neural regeneration after cerebral I/R and the related mechanism. METHODS: Focal cerebral I/R was induced in rats by transient middle cerebral artery occlusion/reperfusion (MCAO/R). The rats were intraperitoneally treated salidroside once daily for 7 consecutive days. Neurobehavioral assessments were performed at 3 days and 7 days after the injury. TTC staining was performed to assess cerebral infarct volume. To evaluate the survival of neurons, immunohistochemical staining of Neuronal Nuclei (NeuN) in the ischemic hemisphere were conducted. Also, immunofluorescence double or triple staining of the biomarkers of proliferating neural progenitor cells in Subventricular Zone (SVZ) and striatum of the ischemia hemisphere were performed to investigate the neurogenesis. Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of neurotrophic factors (NTFs) brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Expression of Notch1 and its target molecular Hes1 were also analyzed by western-blotting and RT-PCR. RESULTS: Salidroside treatment ameliorated I/R induced neurobehavioral impairment, and reduced infarct volume. Salidroside also restored NeuN positive cells loss after I/R injury. Cerebral I/R injury significantly increased the expression of 5-Bromo-2'-Deoxyuridine (BrdU) and doublecotin (DCX), elevated the number of BrdU/Nestin/DCX triple-labeled cells in SVZ, and BrdU/Nestin/glial fibrillary acidic protein (GFAP) triple-labeled cells in striatum. Salidroside treatment further promoted the proliferation of BrdU/DCX labeled neuroblasts and BrdU/Nestin/GFAP labeled reactive astrocytes. Furthermore, salidroside elevated the mRNA expression and protein concentration of BDNF and NGF in ischemia periphery area, as well. Mechanistically, salidroside elevated Notch1/Hes1 mRNA expression in SVZ. The protein levels of them were also increased after salidroside administration. CONCLUSIONS: Salidroside enhances the endogenous neural regeneration after cerebral I/R. The mechanism of the effect may involve the regulation of BDNF/NGF and Notch signaling pathway.
Assuntos
Isquemia Encefálica , Glucosídeos , Regeneração Nervosa , Fenóis , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Transdução de Sinais , Animais , Glucosídeos/farmacologia , Fenóis/farmacologia , Ratos , Masculino , Transdução de Sinais/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Isquemia Encefálica/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fatores de Crescimento Neural/metabolismo , Modelos Animais de Doenças , Receptores Notch/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Neurogênese/efeitos dos fármacosRESUMO
As part of the central nervous system, the optic nerve, composed of axons from retinal ganglion cells (RGCs), generally fails to regenerate on its own when injured in adult mammals. An innovative approach to promoting optic nerve regeneration involves manipulating the interactions between amacrine cells (ACs) and RGCs. Here, we identified a unique AC subtype, dopaminergic ACs (DACs), that responded early after optic nerve crush by down-regulating neuronal activity and reducing retinal dopamine (DA) release. Activating DACs or augmenting DA release with levodopa demonstrated neuroprotective effects and modestly enhanced axon regeneration. Within this context, we pinpointed the DA receptor D1 (DRD1) as a critical mediator of DAC-derived DA and showed that RGC-specific Drd1 overexpression effectively overcame subtype-specific barriers to regeneration. This strategy markedly boosted RGC survival and axon regeneration after crush and preserved vision in a glaucoma model. This study unveils the crucial role of DAC-derived DA signaling in optic nerve regeneration, holding promise for therapeutic insights into neural repair.
Assuntos
Células Amácrinas , Dopamina , Regeneração Nervosa , Nervo Óptico , Células Ganglionares da Retina , Transdução de Sinais , Animais , Células Amácrinas/metabolismo , Dopamina/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Nervo Óptico/metabolismo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/tratamento farmacológico , Traumatismos do Nervo Óptico/patologia , Camundongos , Axônios/metabolismo , Axônios/fisiologia , Receptores de Dopamina D1/metabolismo , Visão Ocular/fisiologia , Modelos Animais de DoençasRESUMO
Peripheral nerve defect are common clinical problem caused by trauma or other diseases, often leading to the loss of sensory and motor function in patients. Autologous nerve transplantation has been the gold standard for repairing peripheral nerve defects, but its clinical application is limited due to insufficient donor tissue. In recent years, the application of tissue engineering methods to synthesize nerve conduits for treating peripheral nerve defect has become a current research focus. This study introduces a novel approach for treating peripheral nerve defects using a tissue-engineered PLCL/SF/NGF@TA-PPy-RGD conduit. The conduit was fabricated by combining electrospun PLCL/SF with an NGF-loaded conductive TA-PPy-RGD gel. The gel, synthesized from RGD-modified tannic acid (TA) and polypyrrole (PPy), provides growth anchor points for nerve cells. In vitro results showed that this hybrid conduit could enhance PC12 cell proliferation, migration, and reduce apoptosis under oxidative stress. Furthermore, the conduit activated the PI3K/AKT signalling pathway in PC12 cells. In a rat model of sciatic nerve defect, the PLCL/SF/NGF@TA-PPy-RGD conduit significantly improved motor function, gastrocnemius muscle function, and myelin sheath axon thickness, comparable to autologous nerve transplantation. It also promoted angiogenesis around the nerve defect. This study suggests that PLCL/SF/NGF@TA-PPy-RGD conduits provide a conducive environment for nerve regeneration, offering a new strategy for peripheral nerve defect treatment, this study provided theoretical basis and new strategies for the research and treatment of peripheral nerve defect.
Assuntos
Hidrogéis , Fator de Crescimento Neural , Regeneração Nervosa , Oligopeptídeos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Nervo Isquiático , Transdução de Sinais , Animais , Regeneração Nervosa/efeitos dos fármacos , Ratos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células PC12 , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Oligopeptídeos/farmacologia , Oligopeptídeos/química , Hidrogéis/química , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/metabolismo , Ratos Sprague-Dawley , Masculino , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Alicerces Teciduais/química , Engenharia Tecidual/métodos , Polímeros/químicaRESUMO
Background: Inhibiting ROS overproduction is considered a very effective strategy for the treatment of peripheral nerve injuries, and Se has a remarkable antioxidant effect; however, since the difference between the effective concentration of Se and the toxic dose is not large, we synthesized a nanomaterial that can release Se slowly so that it can be used more effectively. Methods: Se@SiO2 NPs were synthesized using a mixture of Cu2-x Se nanocrystals, and the mechanism of action of Se@SiO2 NPs was initially explored by performing sequencing, immunofluorescence staining and Western blotting of cellular experiments. The mechanism of action of Se@SiO2 NPs was further determined by performing behavioral assays after animal experiments and by sampling the material for histological staining, immunofluorescence staining, and ELISA. The effects, mechanisms and biocompatibility of Se@SiO2 NPs for peripheral nerve regeneration were determined. Results: Porous Se@SiO2 was successfully synthesized, had good particle properties, and could release Se slowly. CCK-8 experiments revealed that the optimal experimental doses were 100 µM H2O2 and 200 µg/mL Se@SiO2, and RNA-seq revealed that porous Se@SiO2 was associated with cell proliferation, apoptosis, and the PI3K/AKT pathway. WB showed that porous Se@SiO2 could increase the expression of cell proliferation antigens (PCNA and S100) and antiapoptotic proteins (Bcl-2), decrease the expression of proapoptotic proteins (Bax), and increase the expression of antioxidative stress proteins (Nrf2, HO-1, and SOD2). EdU cell proliferation and ROS fluorescence assays showed that porous Se@SiO2 promoted cell proliferation and reduced ROS levels. The therapeutic effect of LY294002 (a PI3K/AKT pathway inhibitor) was decreased significantly and its effect was lost when it was added simultaneously with porous Se@SiO2. Animal experiments revealed that the regenerated nerve fiber density, myelin thickness, axon area, gastrocnemius muscle wet-to-weight ratio, myofiber area, sciatic nerve function index (SFI), CMAP, apoptotic cell ratio, and levels of antioxidative stress proteins and anti-inflammatory factors were increased following the administration of porous Se@SiO2. The levels of oxidative stress proteins and anti-inflammatory factors were significantly greater in the Se@SiO2 group than in the PNI group, and the effect of LY294002 was decreased significantly and was lost when it was added simultaneously with porous Se@SiO2. Conclusion: Se@SiO2 NPs are promising, economical and effective Se-releasing nanomaterials that can effectively reduce ROS production, inhibit apoptosis and promote cell proliferation after nerve injury via the PI3K/AKT pathway, ultimately accelerating nerve regeneration. These findings could be used to design new, promising drugs for the treatment of peripheral nerve injury.
Assuntos
Proliferação de Células , Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Selênio , Transdução de Sinais , Dióxido de Silício , Animais , Selênio/química , Selênio/farmacologia , Dióxido de Silício/química , Dióxido de Silício/farmacologia , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ratos , Apoptose/efeitos dos fármacos , Antioxidantes/farmacologia , Antioxidantes/química , Nanopartículas/química , Masculino , Preparações de Ação Retardada/farmacologia , Preparações de Ação Retardada/química , Ratos Sprague-Dawley , Estresse Oxidativo/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismoRESUMO
Regeneration of sensory axons after a burn injury depends on early keratinocyte responses regulated by the wound microenvironment.
Assuntos
Axônios , Queimaduras , Regeneração Nervosa , Axônios/fisiologia , Regeneração Nervosa/fisiologia , Animais , Humanos , Queratinócitos/fisiologia , Cicatrização/fisiologiaRESUMO
INTRODUCTION: Peripheral nerve injury (PNI) occurs in approximately 3% of all trauma patients and can be challenging to treat, particularly when injury is severe such as with a long-segmental gap. Although peripheral nerves can regenerate after injury, functional recovery is often insufficient, leading to deficits in the quality of life of patients with PNI. Although nerve autografts are the gold standard of care, there are several disadvantages to their use, namely a lack of autologous nerve material for repair. This has led to the pursuit of alternative treatment methods such as axon guidance channels (AGCs). Second-generation AGCs have been shown to be able to deliver growth-enhancing substrates for nerve repair directly to the injury site. Although our laboratory has had success with second-generation AGCs filled with Schwann cells (SCs), SCs have their own set of issues clinically. Because of this, we have begun to utilize SC-derived exosomes as an alternative, as they have the appropriate protein markers, associate to axons in high concentrations, and are able to improve nerve regeneration. However, it is unknown how SC-derived exosomes may react within second-generation AGCs; thus, the aim of the present study was to assess the ability of SC-derived exosomes to be loaded into a second-generation AGC and how they would distribute within it. MATERIALS AND METHODS: A total of 4 dry second-generation AGCs were loaded with SC-derived exosomes that were derived from green fluorescent protein (GFP)-labeled SCs. They were subsequently frozen and sliced before imaging. RESULTS: Here, we present findings that SC-derived exosomes can be loaded into second-generation AGCs through our established loading method utilizing negative pressure and are able to survive and equally distribute along the length of the AGC. CONCLUSIONS: Although only 4 second-generation AGCs were utilized, these findings indicate a potential use for SC-derived exosomes within second-generation AGCs to treat severe PNI. Future research should focus on exploring this in greater detail and in different contexts to assess the ability of SC-derived exosomes to survive at the site of injury and treat PNI.
Assuntos
Exossomos , Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Células de Schwann , Células de Schwann/fisiologia , Regeneração Nervosa/fisiologia , Animais , Traumatismos dos Nervos Periféricos/terapia , Ratos , Orientação de Axônios/fisiologia , Axônios/fisiologiaRESUMO
OBJECTIVES: This study aims to evaluate the histopathological, biochemical, and functional effects of N-acetylcysteine (NAC), which has antioxidant, anti-inflammatory, and cytoprotective activity, on nerve regeneration in rats with sciatic nerve crush (axonotmesis) injury. MATERIALS AND METHODS: This study used 16 male Wistar rats, which were divided into treatment and control groups. A standard axonotmesis-type surgical injury was induced in the left sciatic nerves of all rats. The treatment group was given 300 mg/kg of intraperitoneal NAC once a day, whereas the control group received an equal volume of saline solution. After conducting gait analyses, the sciatic functional index (SFI) was used for functional assessment. After gait analysis, all animals were euthanized. Blood samples were examined biochemically. The left sciatic nerves and left triceps surae muscles were examined histopathologically. RESULTS: Histopathologically, the thickness of the perineurium, axonal degeneration, axonolysis, edema, inflammation, muscle atrophy, and muscle degeneration were all significantly lower in the treatment group (p<0.05). Functionally, SFI-1, SFI-2, and SFI-3 were significantly higher in the treatment group (p<0.05). Biochemically, while the native thiol level and native thiol/total thiol ratio were significantly higher in the treatment group (p<0.003), the disulfide/total thiol ratio was significantly higher in the control group (p<0.005). Significant correlations were found between six of the seven gait parameters and the histopathological findings (p<0.05). CONCLUSION: Our study results suggest that NAC may contribute positively to the histopathological and functional recovery of sciatic nerve injury in rats. Furthermore, NAC may have an antioxidant effect on thiol-disulfide homeostasis at a biochemical level. We believe that NAC has a stimulatory effect on healing following nerve injuries.
Assuntos
Acetilcisteína , Regeneração Nervosa , Ratos Wistar , Nervo Isquiático , Animais , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Masculino , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologia , Nervo Isquiático/lesões , Regeneração Nervosa/efeitos dos fármacos , Ratos , Antioxidantes/farmacologia , Modelos Animais de Doenças , Cicatrização/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Traumatismos dos Nervos Periféricos/patologia , Recuperação de Função Fisiológica/efeitos dos fármacosRESUMO
Spinal cord injuries cause irreversible loss of sensory and motor functions. In mammals, intrinsic and extrinsic inhibitions of neuronal regeneration obstruct neural repair after spinal cord injury. Although astrocytes have been involved in a growing list of vital homeostatic functions in the nervous system, their roles after injury have fascinated and puzzled scientists for decades. Astrocytes undergo long-lasting morphological and functional changes after injury, referred to as reactive astrogliosis. Although reactive astrogliosis is required to contain spinal cord lesions and restore the blood-spinal cord barrier, reactive astrocytes have detrimental effects that inhibit neuronal repair and remyelination. Intriguingly, elevated regenerative capacity is preserved in some non-mammalian vertebrates, where astrocyte-like glial cells display exclusively pro-regenerative effects after injury. A detailed molecular and phenotypic catalog of the continuum of astrocyte reactivity states is an essential first step toward the development of glial cell manipulations for spinal cord repair.
Assuntos
Astrócitos , Neurônios , Traumatismos da Medula Espinal , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/patologia , Astrócitos/metabolismo , Animais , Humanos , Neurônios/metabolismo , Gliose/metabolismo , Gliose/patologia , Regeneração Nervosa/fisiologia , Medula Espinal/metabolismo , Comunicação Celular/fisiologiaRESUMO
Peripheral nerve injury is a major societal concern. Black phosphorus (BP) has inherent advantages over cell-based therapies in regenerative medicine. However, controlling spontaneous degradation and size-dependent cytotoxicity remains challenging and poses difficulties for clinical translation. In this study, we constructed zero-dimensional BP quantum dots (QDs) modified with antioxidant ß-carotene and comprehensively investigated them in Schwann cells (SCs) to elucidate their potential for peripheral nerve repair. In vitro experiments demonstrated that BPQD@ß-carotene has an inappreciable toxicity and good biocompatibility, favoring neural regrowth, angiogenesis, and inflammatory regulation of SCs. Furthermore, the PI3K/Akt and Ras/ERK1/2 signaling pathways were activated in SCs at the genetic, protein, and metabolite levels. The BPQD@ß-carotene-embedded GelMA/PEGDA scaffold enhanced functional recovery by promoting axon remyelination and regeneration and facilitating intraneural angiogenesis in peripheral nerve injury models of rats and beagle dogs. These results contribute to advancing knowledge of BP nanomaterials in tissue regeneration and show significant potential for application in translational medicine.
Assuntos
Antioxidantes , Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Fósforo , Pontos Quânticos , Ratos Sprague-Dawley , Células de Schwann , Animais , Regeneração Nervosa/efeitos dos fármacos , Pontos Quânticos/química , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Traumatismos dos Nervos Periféricos/patologia , Antioxidantes/farmacologia , Antioxidantes/química , Ratos , Cães , Fósforo/química , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Masculino , Células CultivadasRESUMO
BACKGROUND: As a common disabling disease, irreversible neuronal death due to spinal cord injury (SCI) is the root cause of functional impairment; however, the capacity for neuronal regeneration in the developing spinal cord tissue is limited. Therefore, there is an urgent need to investigate how defective neurons can be replenished and functionally integrated by neural regeneration; the reprogramming of intrinsic cells into functional neurons may represent an ideal solution. METHODS: A mouse model of transection SCI was prepared by forceps clamping, and an adeno-associated virus (AAV) carrying the transcription factors NeuroD1 and Neurogenin-2(Ngn2) was injected in situ into the spinal cord to specifically overexpress these transcription factors in astrocytes close to the injury site. 5-bromo-2´-deoxyuridine (BrdU) was subsequently injected intraperitoneally to continuously track cell regeneration, neuroblasts and immature neurons marker expression, neuronal regeneration, and glial scar regeneration. In addition, immunoprotein blotting was used to measure the levels of transforming growth factor-ß (TGF-ß) pathway-related protein expression. We also evaluated motor function, sensory function, and the integrity of the blood-spinal cord barrier(BSCB). RESULTS: The in situ overexpression of NeuroD1 and Ngn2 in the spinal cord was achieved by specific AAV vectors. This intervention led to a significant increase in cell regeneration and the proportion of cells with neuroblasts and immature neurons cell properties at the injury site(p < 0.0001). Immunofluorescence staining identified astrocytes with neuroblasts and immature neurons cell properties at the site of injury while neuronal marker-specific staining revealed an increased number of mature astrocytes at the injury site. Behavioral assessments showed that the intervention did not improve The BMS (Basso mouse scale) score (p = 0.0726) and gait (p > 0.05), although the treated mice had more sensory sensitivity and greater voluntary motor ability in open field than the non-intervention mice. We observed significant repair of the BSCB at the center of the injury site (p < 0.0001) and a significant improvement in glial scar proliferation. Electrophysiological assessments revealed a significant improvement in spinal nerve conduction (p < 0.0001) while immunostaining revealed that the levels of TGF-ß protein at the site of injury in the intervention group were lower than control group (p = 0.0034); in addition, P70 s6 and PP2A related to the TGF-ß pathway showed ascending trend (p = 0.0036, p = 0.0152 respectively). CONCLUSIONS: The in situ overexpression of NeuroD1 and Ngn2 in the spinal cord after spinal cord injury can reprogram astrocytes into neurons and significantly enhance cell regeneration at the injury site. The reprogramming of astrocytes can lead to tissue repair, thus improving the reduced threshold and increasing voluntary movements. This strategy can also improve the integrity of the blood-spinal cord barrier and enhance nerve conduction function. However, the simple reprogramming of astrocytes cannot lead to significant improvements in the striding function of the lower limbs.
Assuntos
Astrócitos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Modelos Animais de Doenças , Proteínas do Tecido Nervoso , Traumatismos da Medula Espinal , Animais , Traumatismos da Medula Espinal/terapia , Traumatismos da Medula Espinal/fisiopatologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Astrócitos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Camundongos , Regeneração Nervosa/fisiologia , Neurônios , Feminino , Camundongos Endogâmicos C57BL , Medula Espinal/metabolismoRESUMO
Damage to the central nervous system (CNS) often leads to irreversible neurological deficits, and there are currently few effective treatments available. However, recent advancements in regenerative medicine have identified CNS organoids as promising therapeutic options for addressing CNS injuries. These organoids, composed of various neurons and supporting cells, have shown potential for direct repair at injury sites. CNS organoids resemble the structure and function of actual brain tissue, which allows them to adapt and function well within the physiological environment when transplanted into injury sites. Research findings suggest that CNS organoids can replace damaged neurons, form new neural connections, and promote neural recovery. This review highlights the emerging benefits, evaluates preclinical transplantation outcomes, and explores future strategies for optimizing neuroregeneration using CNS organoids. With continued research and technological advancements, these organoids could provide new hope for patients suffering from neurological deficits.
Assuntos
Sistema Nervoso Central , Organoides , Humanos , Organoides/citologia , Organoides/transplante , Regeneração Nervosa , Animais , Neurônios/citologia , Neurônios/fisiologia , Medicina Regenerativa/métodosRESUMO
The therapy of large defects in peripheral nerve injury (PNI) suffers from several drawbacks, especially the lack of autologous nerve donors. Nerve conduits are considered as a solution for nerve injury treatment, but biocompatibility improvements is still required for conduits prepared with synthetic materials. Cell-derived extracellular matrix (ECM) has drawn attention due to its lower risk of immunogenic response and independence from donor availability. The goal of this study is to coat bone mesenchymal stem cell-derived ECMs on poly(lactic-co-glycolic) acid (PLGA) conduits to enhance their ability to support neural growth and neurite extensions. The ECM-coated conduits have better hydrophilic properties than the pure PLGA conduits. A marked increase on PC12 and RSC96 cells' viability, proliferation and dorsal root ganglion neurite extension was observed. Quantitative PCR analysis exhibited a significant increase in markers for cell proliferation (GAP43), neurite extension (NF-H, MAP2, andßIII-tubulin) and neural function (TREK-1). These results show the potential of ECM-coated PLGA conduits in PNI therapy.
Assuntos
Proliferação de Células , Sobrevivência Celular , Matriz Extracelular , Células-Tronco Mesenquimais , Regeneração Nervosa , Neuritos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Animais , Ratos , Neuritos/metabolismo , Células PC12 , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Regeneração Nervosa/efeitos dos fármacos , Alicerces Teciduais/química , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Gânglios Espinais , Traumatismos dos Nervos Periféricos/terapia , Engenharia Tecidual/métodos , Polímeros/química , Teste de MateriaisRESUMO
The cell intrinsic mechanisms directing peripheral nerve regeneration have remained largely understudied, thus limiting our understanding of these processes and constraining the advancement of novel clinical therapeutics. The use of primary adult rat dorsal root ganglion (DRG) neurons cultured in vitro is well established. Despite this, these cells can be challenging to culture and have so far not been amenable to robust transfection or live-cell imaging. The ability to transfect these cells with fluorescent plasmid constructs to label subcellular structures, combined with high resolution time-lapse imaging has the potential to provide invaluable insight into how peripheral neurons coordinate their regenerative response, and which specific cellular structures are involved in this process. Here we describe a protocol that facilitates transfection and subsequent live-imaging of adult rat DRG neurons.
Assuntos
Gânglios Espinais , Regeneração Nervosa , Neurônios , Animais , Gânglios Espinais/citologia , Regeneração Nervosa/fisiologia , Ratos , Neurônios/citologia , Neurônios/fisiologia , Neurônios/metabolismo , Células Cultivadas , Transfecção/métodos , Imagem com Lapso de Tempo/métodosRESUMO
The analysis of nerve regeneration in the chemotherapy-induced peripheral neuropathy (CIPN) model can be achieved using the compartmentalized culture system. This system enables us to isolate the cell body from the axon physically and fluidically, therefore allowing for the independent manipulation of the cell body and axons. Compartmentalized chambers mimic the human body conditions, and can be used to study axonal degeneration, disease modeling, and drug screening. This culture system is applied to the CIPN model to study and analyze axonal behavior in response to paclitaxel (PTX) with and without fluocinolone acetonide (FA) and to better understand the site-specific target of PTX. Therefore, this compartmentalized system allows for the independent treatment of chemotherapy drugs to the cell body or axonal side which enables monitoring their reaction as a result of the treatment.
Assuntos
Regeneração Nervosa , Paclitaxel , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Paclitaxel/farmacologia , Animais , Humanos , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Axônios/fisiologia , Axônios/efeitos dos fármacos , Axônios/metabolismo , RatosRESUMO
Isolation and culture of dorsal root ganglion (DRG) neurons from adult animals is a useful experimental system for evaluating neural plasticity after axonal injury, as well as the neurological dysfunction resulting from aging and various types of disease. In this chapter, we will introduce a detailed method for the culture of mature rat DRG neurons. About 30-40 ganglia are dissected from a rat and mechanically and enzymatically digested. Subsequently, density gradient centrifugation of the digested tissue using 30% Percoll efficiently eliminates myelin debris and non-neuronal cells, to afford neuronal cells with a high yield and purity.
Assuntos
Técnicas de Cultura de Células , Separação Celular , Gânglios Espinais , Regeneração Nervosa , Neurônios , Animais , Gânglios Espinais/citologia , Ratos , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Cultura de Células/métodos , Regeneração Nervosa/fisiologia , Separação Celular/métodos , Degeneração Neural/patologia , Células Cultivadas , Centrifugação com Gradiente de Concentração/métodosRESUMO
Axonal damage is a common feature of traumatic injury and neurodegenerative disease. The capacity for axons to regenerate and to recover functionality after injury is a phenomenon that is seen readily in the peripheral nervous system, especially in rodent models, but human axonal regeneration is limited and does not lead to full functional recovery. Here we describe a system where dynamics of human axonal outgrowth and regeneration can be evaluated via live imaging of human-induced pluripotent stem cell (hiPSC)-derived neurons cultured in microfluidic systems, in which cell bodies are isolated from their axons. This system could aid in studying axonal outgrowth dynamics and could be useful for testing potential drugs that encourage regeneration and repair of the nervous system.
Assuntos
Axônios , Células-Tronco Pluripotentes Induzidas , Neurônios Motores , Regeneração Nervosa , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Axônios/fisiologia , Neurônios Motores/fisiologia , Neurônios Motores/citologia , Regeneração Nervosa/fisiologia , Microfluídica/métodos , Microfluídica/instrumentação , Diferenciação Celular , Células Cultivadas , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Cultura de Células/métodosRESUMO
Peripheral nerve injury (PNI) often leads to retrograde cell death in the spinal cord and dorsal root ganglia (DRG), hindering nerve regeneration and functional recovery. Repetitive magnetic stimulation (rMS) promotes nerve regeneration following PNI. Therefore, this study aimed to investigate the effects of rMS on post-injury neuronal death and nerve regeneration. Seventy-two rats underwent autologous sciatic nerve grafting and were divided into two groups: the rMS group, which received rMS and the control (CON) group, which received no treatment. Motor neuron, DRG neuron, and caspase-3 positive DRG neuron counts, as well as DRG mRNA expression analyses, were conducted at 1-, 4-, and 8-weeks post-injury. Functional and axon regeneration analyses were performed at 8-weeks post-injury. The CON group demonstrated a decreased DRG neuron count starting from 1 week post-injury, whereas the rMS group exhibited significantly higher DRG neuron counts at 1- and 4-weeks post-injury. At 8-weeks post-injury, the rMS group demonstrated a significantly greater myelinated nerve fiber density in autografted nerves. Furthermore, functional analysis showed significant improvements in latency and toe angle in the rMS group. Overall, these results suggest that rMS can prevent DRG neuron death and enhance nerve regeneration and motor function recovery after PNI.
Assuntos
Morte Celular , Modelos Animais de Doenças , Gânglios Espinais , Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Nervo Isquiático , Animais , Gânglios Espinais/metabolismo , Ratos , Nervo Isquiático/lesões , Traumatismos dos Nervos Periféricos/terapia , Masculino , Ratos Sprague-Dawley , Neurônios/metabolismo , Magnetoterapia/métodos , Recuperação de Função Fisiológica , Neurônios Motores/metabolismo , Neurônios Motores/fisiologiaRESUMO
Spinal cord injury (SCI) presents a critical medical challenge, marked by substantial neural damage and persistent functional deficits. This study investigates the therapeutic potential of cold atmospheric plasma (CAP) for SCI, utilizing a tailored dielectric barrier discharge (DBD) device to conduct comprehensive in vivo and in vitro analyses. The findings show that CAP treatment significantly improves functional recovery after SCI, reduces neuronal apoptosis, lowers inflammation, and increases axonal regeneration. These findings illustrate the efficacy of CAP in fostering a conducive environment for recovery by modulating inflammatory responses, enhancing neuronal survival, and encouraging regenerative processes. The underlying mechanism involves CAP's reactive oxygen species (ROS) reduction, followed by activating antioxidant enzymes. These findings position CAP as a pioneering approach for spinal cord injury (SCI) treatment, presenting opportunities for improved neural recovery and establishing a new paradigm in SCI therapy.
Assuntos
Estresse Oxidativo , Espécies Reativas de Oxigênio , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Recuperação de Função Fisiológica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Gases em Plasma/farmacologia , Gases em Plasma/uso terapêutico , Feminino , Ratos , Regeneração Nervosa/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Modelos Animais de DoençasRESUMO
Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here, we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory balance after injury. In addition, a transient population of injury-responsive neurons (iNeurons) show elevated plasticity 1 week post-injury. We found iNeurons are injury-surviving neurons that acquire a neuroblast-like gene expression signature after injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity-driven neural repair.
Assuntos
Neurogênese , Plasticidade Neuronal , Análise de Célula Única , Traumatismos da Medula Espinal , Regeneração da Medula Espinal , Medula Espinal , Peixe-Zebra , Animais , Traumatismos da Medula Espinal/metabolismo , Plasticidade Neuronal/fisiologia , Neurogênese/genética , Medula Espinal/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Sistemas CRISPR-Cas , Neurônios GABAérgicos/metabolismo , Recuperação de Função Fisiológica , Modelos Animais de Doenças , Regeneração Nervosa/fisiologia , Animais Geneticamente ModificadosRESUMO
BACKGROUND: Zebrafish are vertebrates with a high potential of regeneration after injury in the central nervous system. Therefore, they have emerged as a useful model system for studying traumatic spinal cord injuries. NEW METHOD: Using larval zebrafish, we have developed a robust paradigm to model the effects of anterior spinal cord injury, which correspond to the debilitating injuries of the cervical and thoracic regions in humans. Our new paradigm consists of a more anterior injury location compared to previous studies, a modified behavioral assessment using the visual motor response, and a new data analysis code. RESULTS: Our approach enables a spinal cord injury closer to the hindbrain with more functional impact compared to previous studies using a more posterior injury location. Results reported in this work reveal recovery over seven days following spinal cord injury. COMPARING WITH EXISTING METHODS: The present work describes a modified paradigm for the in vivo study of spinal cord regeneration after injury using larval zebrafish, including an anterior injury location, a robust behavioral assessment, and a new data analysis software. CONCLUSIONS: Our findings lay the foundation for applying this paradigm to study the effects of drugs, nutrition, and other treatments to improve the regeneration process.