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Mitophagy selectively eliminates damaged or dysfunctional mitochondria, playing a crucial role in maintaining mitochondrial quality control. However, it remains unclear whether mitophagy can be fully activated and how it evolves after SCI. Our RNA-seq analysis of animal samples from sham and 1, 3, 5, and 7 days post-SCI indicated that mitophagy was indeed inhibited during the acute and subacute early stages. In vitro experiments showed that this inhibition was closely related to excessive production of reactive oxygen species (ROS) and the downregulation of BNIP3. Excessive ROS led to the blockage of mitophagy flux, accompanied by further mitochondrial dysfunction and increased neuronal apoptosis. Fortunately, ligustilide (LIG) was found to have the ability to reverse the oxidative stress-induced downregulation of BNIP3 and enhance mitophagy through BNIP3-LC3 interaction, alleviating mitochondrial dysfunction and ultimately reducing neuronal apoptosis. Further animal experiments demonstrated that LIG alleviated oxidative stress and mitophagy inhibition, rescued neuronal apoptosis, and promoted tissue repair, ultimately leading to improved motor function. In summary, this study elucidated the state of mitophagy inhibition following SCI and its potential mechanisms, and confirmed the effects of LIG-enhanced mitophagy through BNIP3-LC3, providing new therapeutic targets and strategies for repairing SCI.
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4-Butirolactona , Apoptosis , Proteínas de la Membrana , Mitofagia , Neuronas , Estrés Oxidativo , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Neuronas/metabolismo , Traumatismos de la Médula Espinal/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Masculino , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Asociadas a MicrotúbulosRESUMEN
BACKGROUND AND OBJECTIVES: Spinal cord injury (SCI) results in significant neurological deficits, and microglia play the critical role in regulating the immune microenvironment and neurological recovery. Protein lactylation has been found to modulate the function of immune cells. Therefore, this study aimed to elucidate the effects of glycolysis-derived lactate on microglial function and its potential neuroprotective mechanisms via lactylation after SCI. METHODS: Single-cell RNA sequencing (scRNA-seq) data were obtained from figshare to analyze cellular and molecular alterations within the spinal cord post-SCI, further focusing on the expression of microglia-related genes for cell sub-clustering, trajectory analysis, and glycolysis function analysis. We also evaluated the expression of lactylation-related genes in microglia between day 7 after SCI and sham group. Additionally, we established the mice SCI model and performed the bulk RNA sequencing in a time-dependent manner. The expression of glycolysis- and lactylation-related genes was evaluated, as well as the immune infiltration analysis based on the lactylation-related genes. Then, we investigated the bio-effects of lactate on the inflammation and polarization phenotype of microglia. Finally, adult male C57BL/6 mice were subjected to exercise first to increase lactate level, before SCI surgery, aiming to evaluate the protective effects of lactate-mediated lactylation of microglia-related proteins on SCI. RESULTS: scRNA-seq identified a subcluster of microglia, recombinant chemokine C-X3-C-motif receptor 1+ (CX3CR1+) microglia, which is featured by M1-like phenotype and increased after SCI. KEGG analysis revealed the dysfunctional glycolysis in microglia after SCI surgery, and AUCell analysis suggested that the decreased glycolysis an increased oxidative phosphorylation in CX3CR1+ microglia. Differential gene analysis suggested that several lactylation-related genes (Fabp5, Lgals1, Vim, and Nefl) were downregulated in CX3CR1+ microglia at day 7 after SCI, further validated by the results from bulk RNA sequencing. Immunofluorescence staining indicated the expression of lactate dehydrogenase A (LDHA) in CX3CR1+ microglia also decreased at day 7 after SCI. Cellular experiments demonstrated that the administration of lactate could increase the lactylation level and inhibit the pro-inflammatory phenotype in microglia. Functionally, exercise-mediated lactate production resulted in improved locomotor recovery and decreased inflammatory markers in SCI mice compared to SCI alone. CONCLUSIONS: In the subacute phase of SCI, metabolic remodeling in microglia may be key therapeutic targets to promote nerve regeneration, and lactate contributed to neuroprotection after SCI by influencing microglial lactylation and inflammatory phenotype, which offered a novel approach for therapeutic intervention.
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Ácido Láctico , Ratones Endogámicos C57BL , Microglía , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Traumatismos de la Médula Espinal , Animales , Traumatismos de la Médula Espinal/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Ratones , Masculino , Ácido Láctico/metabolismo , Análisis de Secuencia de ARN/métodos , Fármacos Neuroprotectores/farmacología , Glucólisis/efectos de los fármacos , Glucólisis/fisiologíaRESUMEN
Traumatic spinal cord injury (tSCI) is a severe injury to the central nervous system that is categorized into primary and secondary injuries. Among them, the local microenvironmental imbalance in the spinal cord caused by secondary spinal cord injury includes accumulation of cytokines and chemokines, reduced angiogenesis, dysregulation of cellular energy metabolism, and dysfunction of immune cells at the site of injury, which severely impedes neurological recovery from spinal cord injury (SCI). In recent years, single-cell techniques have revealed the heterogeneity of multiple immune cells at the genomic, transcriptomic, proteomic, and metabolomic levels after tSCI, further deepening our understanding of the mechanisms underlying tSCI. However, spatial information about the tSCI microenvironment, such as cell location and cell-cell interactions, is lost in these approaches. The application of spatial multi-omics technology can solve this problem by combining the data obtained from immunohistochemistry and multiparametric analysis to reveal the changes in the microenvironment at different times of secondary injury after SCI. In this review, we systematically review the progress of spatial multi-omics techniques in the study of the microenvironment after SCI, including changes in the immune microenvironment and discuss potential future therapeutic strategies.
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Microambiente Celular , Proteómica , Traumatismos de la Médula Espinal , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/metabolismo , Humanos , Microambiente Celular/inmunología , Proteómica/métodos , Animales , Metabolómica/métodos , Genómica/métodos , Transcriptoma , Análisis de la Célula Individual , MultiómicaRESUMEN
AIM: We aimed to explore whether the combination of CLP290 and bumetanide maximally improves neuropathic pain following spinal cord injury (SCI) and its possible molecular mechanism. METHODS: Rats were randomly divided into five groups: Sham, SCI + vehicle, SCI + CLP290, SCI + bumetanide, and SCI + combination (CLP290 + bumetanide). Drug administration commenced on the 7th day post-injury (7 dpi) and continued for 14 days. All rats underwent behavioral assessments for 56 days to comprehensively evaluate the effects of interventions on mechanical pain, thermal pain, cold pain, motor function, and other relevant parameters. Electrophysiological assessments, immunoblotting, and immunofluorescence detection were performed at different timepoints post-injury, with a specific focus on the expression and changes of KCC2 and NKCC1 proteins in the lumbar enlargement of the spinal cord. RESULTS: CLP290 and bumetanide alleviated SCI-associated hypersensitivity and locomotor function, with the combination providing enhanced recovery. The combined treatment group exhibited the most significant improvement in restoring Rate-Dependent Depression (RDD) levels. In the combined treatment group and the two individual drug administration groups, the upregulation of potassium chloride cotransporter 2 (K+-Cl-cotransporter 2, KCC2) expression and downregulation of sodium potassium chloride cotransporter 1 (Na+-K+-Cl-cotransporter 1, NKCC1) expression in the lumbar enlargement area resulted in a significant increase in the KCC2/NKCC1 ratio compared to the SCI + vehicle group, with the most pronounced improvement seen in the combined treatment group. Compared to the SCI + vehicle group, the SCI + bumetanide group showed no significant paw withdrawal thermal latency (PWTL) improvement at 21 and 35 dpi, but a notable enhancement at 56 dpi. In contrast, the SCI + CLP290 group significantly improved PWTL at 21 days, with non-significant changes at 35 and 56 days. At 21 dpi, KCC2 expression was marginally higher in monotherapy groups versus SCI + vehicle, but not significantly. At 56 dpi, only the SCI + bumetanide group showed a significant difference in KCC2 expression compared to the control group. CONCLUSION: Combined application of CLP290 and bumetanide effectively increases the ratio of KCC2/NKCC1, restores RDD levels, enhances GABAA receptor-mediated inhibitory function in the spinal cord, and relieves neuropathic pain in SCI; Bumetanide significantly improves neuropathic pain in the long term, whereas CLP290 demonstrates a notable short-term effect.
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Bumetanida , Cotransportadores de K Cl , Neuralgia , Ratas Sprague-Dawley , Miembro 2 de la Familia de Transportadores de Soluto 12 , Traumatismos de la Médula Espinal , Simportadores , Animales , Bumetanida/farmacología , Bumetanida/uso terapéutico , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/metabolismo , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Neuralgia/metabolismo , Ratas , Masculino , Simportadores/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/uso terapéutico , Quimioterapia Combinada , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Acetatos , IndenosRESUMEN
OBJECTIVE: To investigate the mechanism underlying the neuroprotective effect of linarin (LIN) against microglia activation-mediated inflammation and neuronal apoptosis following spinal cord injury (SCI). METHODS: Fifty C57BL/6J mice (8- 10 weeks old) were randomized to receive sham operation, SCI and linarin treatment at 12.5, 25, and 50 mg/kg following SCI (n=10). Locomotor function recovery of the SCI mice was assessed using the Basso Mouse Scale, inclined plane test, and footprint analysis, and spinal cord tissue damage and myelination were evaluated using HE and LFB staining. Nissl staining, immunofluorescence assay and Western blotting were used to observe surviving anterior horn motor neurons in injured spinal cord tissue. In cultured BV2 cells, the effects of linarin against lipopolysaccharide (LPS)induced microglia activation, inflammatory factor release and signaling pathway changes were assessed with immunofluorescence staining, Western blotting, RT-qPCR, and ELISA. In a BV2 and HT22 cell co-culture system, Western blotting was performed to examine the effect of linarin against HT22 cell apoptosis mediated by LPS-induced microglia activation. RESULTS: Linarin treatment significantly improved locomotor function (P < 0.05), reduced spinal cord damage area, increased spinal cord myelination, and increased the number of motor neurons in the anterior horn of the SCI mice (P < 0.05). In both SCI mice and cultured BV2 cells, linarin effectively inhibited glial cell activation and suppressed the release of iNOS, COX-2, TNF-α, IL-6, and IL-1ß, resulting also in reduced neuronal apoptosis in SCI mice (P < 0.05). Western blotting suggested that linarin-induced microglial activation inhibition was mediated by inhibition of the TLR4/NF- κB signaling pathway. In the cell co-culture experiments, linarin treatment significantly decreased inflammation-mediated apoptosis of HT22 cells (P < 0.05). CONCLUSION: The neuroprotective effect of linarin is medicated by inhibition of microglia activation via suppressing the TLR4/NFκB signaling pathway, which mitigates neural inflammation and reduce neuronal apoptosis to enhance motor function of the SCI mice.
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Apoptosis , Ratones Endogámicos C57BL , Microglía , FN-kappa B , Transducción de Señal , Traumatismos de la Médula Espinal , Receptor Toll-Like 4 , Animales , Ratones , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Receptor Toll-Like 4/metabolismo , Apoptosis/efectos de los fármacos , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Cumarinas/farmacología , Inflamación/metabolismo , Lipopolisacáridos , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/etiología , GlicósidosRESUMEN
The pathological cascade of spinal cord injury (SCI) is highly intricate. The onset of neuroinflammation can exacerbate the extent of damage. Pyroptosis is a form of inflammation-linked programmed cell death (PCD), the inhibition of pyroptosis can partially mitigate neuroinflammation. It is imperative to delineate the principal cell types susceptible to pyroptosis and concomitantly identify key genes associated with this process. We initially defined the pyroptosis-related genes (PRGs) and analyzed their expression at different time points post SCI. The results demonstrate a substantial upregulation of differentially expressed genes (DEGs) related to pyroptosis on the 7 days post-injury (dpi), these DEGs in the 7 dpi are closely related to the inflammatory response. Subsequently, immune infiltration analysis revealed a predominant presence of inflammatory microglia. Through correlation analysis, we postulated that pyroptosis primarily manifested within the inflammatory microglia. Employing machine learning algorithms, we identified four pyroptosis-related molecular signatures, which were experimentally validated using BV2 cells and spinal cord tissue samples. The robustness of the identified molecular signatures was further confirmed through single-cell sequencing data analysis. Overall, our study elucidates the temporal dynamics of pyroptosis and identifies key molecular signatures following SCI. These findings can provide novel evidence for therapeutic interventions in SCI.
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Aprendizaje Automático , Microglía , Piroptosis , Análisis de la Célula Individual , Traumatismos de la Médula Espinal , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/metabolismo , Análisis de la Célula Individual/métodos , Animales , Microglía/metabolismo , Ratones , Médula Espinal/metabolismo , Médula Espinal/patología , Ratones Endogámicos C57BL , Masculino , Enfermedades Neuroinflamatorias/genética , Línea Celular , Modelos Animales de EnfermedadRESUMEN
Understanding the sequence of cellular responses and their contributions to pathomorphogical changes in spinal white matter injuries is a prerequisite for developing efficient therapeutic strategies for spinal cord injury (SCI) as well as neurodegenerative and inflammatory diseases of the spinal cord such as amyotrophic lateral sclerosis and multiple sclerosis. We have developed several types of surgical procedures suitable for acute one-time and chronic recurrent in vivo multiphoton microscopy of spinal white matter [1]. Sophisticated surgical procedures were combined with transgenic mouse technology to image spinal tissue labeled with up to four fluorescent proteins (FPs) in axons, astrocytes, microglia, and blood vessels. To clearly separate the simultaneously excited FPs, spectral unmixing including iterative procedures was performed after imaging the diversely labeled spinal white matter with a custom-made 4-channel two-photon laser-scanning microscope. In our longitudinal multicellular studies of injured spinal white matter, we imaged axonal dynamics and invasion of microglia and astrocytes for a time course of over 200 days after SCI. Our methods offer ideal platforms for investigating acute and chronic cellular dynamics, cell-cell interactions, and metabolite fluctuations in health and disease as well as pharmacological manipulations in vivo.
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Axones , Ratones Transgénicos , Traumatismos de la Médula Espinal , Sustancia Blanca , Animales , Sustancia Blanca/patología , Sustancia Blanca/metabolismo , Sustancia Blanca/diagnóstico por imagen , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/diagnóstico por imagen , Axones/patología , Axones/metabolismo , Neuroglía/metabolismo , Neuroglía/patología , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Médula Espinal/patología , Médula Espinal/metabolismo , Microglía/metabolismo , Microglía/patología , Astrocitos/metabolismo , Astrocitos/patologíaRESUMEN
Traumatic spinal cord injury (tSCI) has complex pathophysiological events that begin after the initial trauma. One such event is fibroglial scar formation by fibroblasts and reactive astrocytes. A strong inhibition of axonal growth is caused by the activated astroglial cells as a component of fibroglial scarring through the production of inhibitory molecules, such as chondroitin sulfate proteoglycans or myelin-associated proteins. Here, we used neural precursor cells (aldynoglia) as promoters of axonal growth and a fibrin hydrogel gelled under alkaline conditions to support and guide neuronal cell growth, respectively. We added Tol-51 sulfoglycolipid as a synthetic inhibitor of astrocyte and microglia in order to test its effect on the axonal growth-promoting function of aldynoglia precursor cells. We obtained an increase in GFAP expression corresponding to the expected glial phenotype for aldynoglia cells cultured in alkaline fibrin. In co-cultures of dorsal root ganglia (DRG) and aldynoglia, the axonal growth promotion of DRG neurons by aldynoglia was not affected. We observed that the neural precursor cells first clustered together and then formed niches from which aldynoglia cells grew and connected to groups of adjacent cells. We conclude that the combination of alkaline fibrin with synthetic sulfoglycolipid Tol-51 increased cell adhesion, cell migration, fasciculation, and axonal growth capacity, promoted by aldynoglia cells. There was no negative effect on the behavior of aldynoglia cells after the addition of sulfoglycolipid Tol-51, suggesting that a combination of aldynoglia plus alkaline fibrin and Tol-51 compound could be useful as a therapeutic strategy for tSCI repair.
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Axones , Fibrina , Ganglios Espinales , Animales , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Axones/metabolismo , Axones/efectos de los fármacos , Fibrina/metabolismo , Hidrogeles/química , Hidrogeles/farmacología , Ratas , Glucolípidos/farmacología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/citología , Movimiento Celular/efectos de los fármacosRESUMEN
Our previous in vitro studies showed that excitotoxicity evoked by glutamate analogue kainate (KA) significantly decreased the number of rat spinal neurons and triggered high release of glutamate leading to locomotor network block. Our current objective was to assess the role of CREB as a predictive marker of damage following chemically-induced spinal cord injury by using in vivo and in vitro models. Thus, in vivo excitotoxicity in Balb/c adult mice was induced by KA intraspinal injection, while in vitro spinal cord excitotoxicity was produced by bath-applied KA. KA application evoked significant neuronal loss, deterioration in hindlimb motor coordination and thermal allodynia. In addition, immunohistochemical analysis showed that KA application resulted in decreased number of CREB positive nuclei in the ventral horn and in dorsal layers III-IV. Our data suggests that excitotoxic-induced neuronal loss may be potentially predicted by altered CREB nuclear translocation.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Ácido Kaínico , Ratones Endogámicos BALB C , Nocicepción , Médula Espinal , Animales , Ácido Kaínico/farmacología , Ratones , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Nocicepción/efectos de los fármacos , Masculino , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Agonistas de Aminoácidos Excitadores/toxicidad , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/inducido químicamente , Locomoción/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
Spinal cord injury (SCI) results in irreversible neurological impairment. After SCI, Ferritinophagy-induced free iron released from ferritin can lead to extensive lipid peroxidation and aggravate neurological damage. NRF2/HO-1 pathway is to endow cells with a protective effect against oxidative stress, and it plays an important role in the transcriptional activation of a series of antioxidant and detoxification genes. UAMC-3203 is a ferrostatin-1(Fer-1) analogue with better solubility and stability, which can more effectively inhibit ferroptosis after SCI. A rat SCI model was constructed, and the recovery of motor function was observed after treatment with UAMC-3203. ELISA was employed to assess the impact of UAMC-3203 on inflammation-related factors, while immunofluorescence was utilized to investigate the influence of UAMC-3203 on neuronal count as well as the activation of astrocytes and microglia/macrophages. Malondialdehyde (MDA) were detected to reflect the level of oxidation products. Western blot analysis was used to measure the level of ferroptosis markers and the expression of NRF2/HO-1. Our findings demonstrate that UAMC-3203 inhibits the production of reactive oxygen species (ROS) and lipid peroxides, preventing ferroptosis and reducing neuronal degeneration. Additionally, UAMC-3203 suppresses astrocyte proliferation and microglia/macrophage activation, as well as the release of ferroptosis-related inflammatory factors. These combined effects contribute to the preservation of spinal cord tissue and the facilitation of motor function recovery. UAMC-3203 maybe inhibit ferroptosis after SCI to promote functional recovery.
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Ferroptosis , Factor 2 Relacionado con NF-E2 , Recuperación de la Función , Traumatismos de la Médula Espinal , Animales , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Ferroptosis/efectos de los fármacos , Ratas , Recuperación de la Función/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Modelos Animales de Enfermedad , Masculino , Ciclohexilaminas/farmacología , Estrés Oxidativo/efectos de los fármacos , Fenilendiaminas/farmacología , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Microglía/metabolismo , Microglía/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)RESUMEN
Recent studies have highlighted the therapeutic potential of stem cells for various diseases. However, unlike other tissues, brain tissue has a specific structure, consisting of synapses. These synapses not only transmit but also process and refine information. Therefore, synaptic regeneration plays a key role in therapy of neurodegenerative disorders. Neurexins (NRXNs) and neuroligins (NLGNs) are synaptic cell adhesion molecules that connect pre- and postsynaptic neurons at synapses, mediate trans-synaptic signaling, and shape neural network properties by specifying synaptic functions. In this study, we investigated the synaptic regeneration effect of human neural stem cells (NSCs) overexpressing NRXNs (F3.NRXN) and NLGNs (F3.NLGN) in a spinal cord injury model. Overexpression of NRXNs and NLGNs in the neural stem cells upregulated the expression of synaptophysin, PSD95, VAMP2, and synapsin, which are synaptic markers. The BMS scores indicated that the transplantation of F3.NRXN and F3.NLGN enhanced the recovery of locomotor function in adult rodents following spinal cord injury. Transplanted F3.NRXN and F3.NLGN differentiated into neurons and formed a synapse with the host cells in the spinal cord injury mouse model. In addition, F3.NRXN and F3.NLGN cells restored growth factors (GFs) and neurotrophic factors (NFs) and induced the proliferation of host cells. This study suggested that NSCs overexpressing NRXNs and NLGNs could be candidates for cell therapy in spinal cord injuries by facilitating synaptic regeneration.
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Moléculas de Adhesión Celular Neuronal , Modelos Animales de Enfermedad , Células-Madre Neurales , Traumatismos de la Médula Espinal , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/terapia , Traumatismos de la Médula Espinal/genética , Células-Madre Neurales/metabolismo , Animales , Humanos , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Ratones , Sinapsis/metabolismo , Trasplante de Células Madre/métodos , Diferenciación Celular , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Femenino , NeuroliginasRESUMEN
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.
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Astrocitos , Neuronas , Traumatismos de la Médula Espinal , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/patología , Astrocitos/metabolismo , Animales , Humanos , Neuronas/metabolismo , Gliosis/metabolismo , Gliosis/patología , Regeneración Nerviosa/fisiología , Médula Espinal/metabolismo , Comunicación Celular/fisiologíaRESUMEN
Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain.
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Analgésicos Opioides , AMP Cíclico , Gabapentina , Neuralgia , Transducción de Señal , Traumatismos de la Médula Espinal , Animales , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , AMP Cíclico/metabolismo , Ratas , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/metabolismo , Analgésicos Opioides/farmacología , Gabapentina/farmacología , Transducción de Señal/efectos de los fármacos , Ratas Sprague-Dawley , Masculino , Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Pregabalina/farmacología , Pregabalina/uso terapéutico , Sinergismo Farmacológico , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
BACKGROUND: The inflammatory response and scar formation after spinal cord injury (SCI) limit nerve regeneration and functional recovery. Our research group has previously shown that the expression of astrocyte-derived lipocalin 2 (Lcn2) is upregulated after SCI, which correlates with neuronal apoptosis and functional recovery. Therefore, we speculate that astrocyte-specific knockdown of Lcn2 after SCI may lead to a better prognosis. METHODS: Tissue RNA sequencing, Western blotting, PCR, and immunofluorescence assays were conducted to assess the expression of Lcn2 following SCI in mice. Adeno-associated virus 9 (AAV9) transfection was employed to specifically reduce the expression of Lcn2 in astrocytes, and subsequent evaluations of scarring and inflammation were conducted. In vitro experiments involved treating primary astrocytes with TGF-ß or an A1-induced mixture (C1q, TNF-α and IL-1α) following Lcn2 knockdown. Finally, the intrathecal injection of recombinant Lcn2 (ReLcn2) protein was conducted post-injury to further confirm the role of Lcn2 and its underlying mechanism in SCI. RESULTS: Lcn2 expression was elevated in astrocytes after SCI at 7 dpi (days post injury). Lcn2 knockdown in astrocytes is beneficial for neuronal survival and functional recovery after SCI, and is accompanied by a reduced inflammatory response and inhibited scar formation. The inhibition of SMAD-associated signaling activation was identified as a possible mechanism, and in vitro experiments further confirmed this finding. ReLcn2 further activated SMAD-associated signaling and aggravated motor function after SCI. CONCLUSION: The upregulation of Lcn2 expression in astrocytes is involved in neuroinflammation and scar formation after SCI, and the activation of SMAD-associated signaling is one of the underlying mechanisms.
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Astrocitos , Cicatriz , Lipocalina 2 , Ratones Endogámicos C57BL , Proteínas Smad , Traumatismos de la Médula Espinal , Animales , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/genética , Lipocalina 2/genética , Lipocalina 2/metabolismo , Ratones , Astrocitos/metabolismo , Cicatriz/etiología , Cicatriz/patología , Cicatriz/metabolismo , Proteínas Smad/metabolismo , Inflamación/metabolismo , Inflamación/patología , Inflamación/etiología , Masculino , Enfermedades Neuroinflamatorias/etiología , Femenino , Recuperación de la Función/fisiología , Células CultivadasRESUMEN
Spinal cord injury (SCI) is one of the most serious health problems, with no effective therapy. Recent studies indicate that Fisetin, a natural polyphenolic flavonoid, exhibits multiple functions, such as life-prolonging, antioxidant, antitumor, and neuroprotection. However, the restorative effects of Fisetin on SCI and the underlying mechanism are still unclear. In the present study, we found that Fisetin reduced LPS-induced apoptosis and oxidative damage in PC12 cells and reversed LPS-induced M1 polarization in BV2 cells. Additionally, Fisetin safely and effectively promoted the motor function recovery of SCI mice by attenuating neurological damage and promoting neurogenesis at the lesion. Moreover, Fisetin administration inhibited glial scar formation, modulated microglia/macrophage polarization, and reduced neuroinflammation. Network pharmacology, RNA-seq, and molecular biology revealed that Fisetin inhibited the activation of the JAK2/STAT3 signaling pathway. Notably, Colivelin TFA, an activator of JAK2/STAT3 signaling, attenuated Fis-mediated neuroinflammation inhibition and therapeutic effects on SCI mice. Collectively, Fisetin promotes functional recovery after SCI by inhibiting microglia/macrophage M1 polarization and the JAK2/STAT3 signaling pathway. Thus, Fisetin may be a promising therapeutic drug for the treatment of SCI.
Asunto(s)
Flavonoles , Janus Quinasa 2 , Macrófagos , Microglía , Factor de Transcripción STAT3 , Transducción de Señal , Traumatismos de la Médula Espinal , Animales , Humanos , Masculino , Ratones , Ratas , Polaridad Celular/efectos de los fármacos , Flavonoides/farmacología , Flavonoides/administración & dosificación , Flavonoles/farmacología , Janus Quinasa 2/metabolismo , Janus Quinasa 2/genética , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/inmunología , Células PC12 , Recuperación de la Función/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/inmunología , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
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.
Asunto(s)
Neurogénesis , Plasticidad Neuronal , Análisis de la Célula Individual , Traumatismos de la Médula Espinal , Regeneración de la Medula Espinal , Médula Espinal , Pez Cebra , Animales , Traumatismos de la Médula Espinal/metabolismo , Plasticidad Neuronal/fisiología , Neurogénesis/genética , Médula Espinal/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Sistemas CRISPR-Cas , Neuronas GABAérgicas/metabolismo , Recuperación de la Función , Modelos Animales de Enfermedad , Regeneración Nerviosa/fisiología , Animales Modificados GenéticamenteRESUMEN
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.
Asunto(s)
Estrés Oxidativo , Especies Reactivas de Oxígeno , Recuperación de la Función , Traumatismos de la Médula Espinal , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/fisiopatología , Estrés Oxidativo/efectos de los fármacos , Animales , Recuperación de la Función/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Gases em Plasma/farmacología , Gases em Plasma/uso terapéutico , Femenino , Ratas , Regeneración Nerviosa/efectos de los fármacos , Apoptosis/efectos de los fármacos , Modelos Animales de EnfermedadRESUMEN
Using the method of electron paramagnetic resonance spectroscopy, we showed that NO production decreases by 60% (p<0.05) in the region located rostral to the spinal cord injury 7 days after combined injury to the brain and spinal cord. At the same time, NO production did not change in the site of spinal cord injury and caudal to the injury. The intensity of NO production in similar parts of the spinal cord in intact animals remained unchanged.
Asunto(s)
Óxido Nítrico , Traumatismos de la Médula Espinal , Médula Espinal , Animales , Óxido Nítrico/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Médula Espinal/metabolismo , Ratas , Espectroscopía de Resonancia por Spin del Electrón , Masculino , Ratas Wistar , Modelos Animales de Enfermedad , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patologíaRESUMEN
Functional and pathological recovery after spinal cord injury (SCI) is often incomplete due to the limited regenerative capacity of the central nervous system (CNS), which is further impaired by several mechanisms that sustain tissue damage. Among these, the chronic activation of immune cells can cause a persistent state of local CNS inflammation and damage. However, the mechanisms that sustain this persistent maladaptive immune response in SCI have not been fully clarified yet. In this study, we integrated histological analyses with proteomic, lipidomic, transcriptomic, and epitranscriptomic approaches to study the pathological and molecular alterations that develop in a mouse model of cervical spinal cord hemicontusion. We found significant pathological alterations of the lesion rim with myelin damage and axonal loss that persisted throughout the late chronic phase of SCI. This was coupled by a progressive lipid accumulation in myeloid cells, including resident microglia and infiltrating monocyte-derived macrophages. At tissue level, we found significant changes of proteins indicative of glycolytic, tricarboxylic acid cycle (TCA), and fatty acid metabolic pathways with an accumulation of triacylglycerides with C16:0 fatty acyl chains in chronic SCI. Following transcriptomic, proteomic, and epitranscriptomic studies identified an increase of cholesterol and m6A methylation in lipid-droplet-accumulating myeloid cells as a core feature of chronic SCI. By characterizing the multiple metabolic pathways altered in SCI, our work highlights a key role of lipid metabolism in the chronic response of the immune and central nervous system to damage.
Asunto(s)
Metabolismo de los Lípidos , Ratones Endogámicos C57BL , Proteómica , Traumatismos de la Médula Espinal , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Animales , Ratones , Metabolismo de los Lípidos/fisiología , Femenino , Lipidómica , Transcriptoma , MultiómicaRESUMEN
BACKGROUND: Inadequate nerve regeneration and an inhibitory local microenvironment are major obstacles to the repair of spinal cord injury (SCI). The activation and differentiation fate regulation of endogenous neural stem cells (NSCs) represent one of the most promising repair approaches. Metformin has been extensively studied for its antioxidative, anti-inflammatory, anti-aging, and autophagy-regulating properties in central nervous system diseases. However, the effects of metformin on endogenous NSCs remains to be elucidated. METHODS: The proliferation and differentiation abilities of NSCs were evaluated using CCK-8 assay, EdU/Ki67 staining and immunofluorescence staining. Changes in the expression of key proteins related to ferroptosis in NSCs were detected using Western Blot and immunofluorescence staining. The levels of reactive oxygen species, glutathione and tissue iron were measured using corresponding assay kits. Changes in mitochondrial morphology and membrane potential were observed using transmission electron microscopy and JC-1 fluorescence probe. Locomotor function recovery after SCI in rats was assessed through BBB score, LSS score, CatWalk gait analysis, and electrophysiological testing. The expression of the AMPK pathway was examined using Western Blot. RESULTS: Metformin promoted the proliferation and neuronal differentiation of NSCs both in vitro and in vivo. Furthermore, a ferroptosis model of NSCs using erastin treatment was established in vitro, and metformin treatment could reverse the changes in the expression of key ferroptosis-related proteins, increase glutathione synthesis, reduce reactive oxygen species production and improve mitochondrial membrane potential and morphology. Moreover, metformin administration improved locomotor function recovery and histological outcomes following SCI in rats. Notably, all the above beneficial effects of metformin were completely abolished upon addition of compound C, a specific inhibitor of AMP-activated protein kinase (AMPK). CONCLUSION: Metformin, driven by canonical AMPK-dependent regulation, promotes proliferation and neuronal differentiation of endogenous NSCs while inhibiting ferroptosis, thereby facilitating recovery of locomotor function following SCI. Our study further elucidates the protective mechanism of metformin in SCI, providing new mechanistic insights for its candidacy as a therapeutic agent for SCI.