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SUMMARY: This study investigated the role and mechanism of aspirin combined with rehabilitation training in the nerve injury repair and Schwann cell changes in rats with sciatic nerve injury. Totally, 120 male healthy SD rats were randomly divided into sham, model, aspirin, and aspirin + rehabilitation groups, with 30 rats in each group. The sciatic nerve function index (SFI), photothermal pain tolerance threshold and inclined plane test results at 4, 6, and 8 weeks after operation were compared. The distance of sensory nerve regeneration and the expression of S100B protein in Schwann cells were analyzed. Compared with the sham group, the SFI of the model, aspirin, and aspirin+rehabilitation groups were significantly lower at 4, 6, and 8 weeks after operation. However, the aspirin and aspirin+rehabilitation groups had significantly higher SFI than the model group. The SFI at 6 and 8 weeks after operation was higher in the aspirin+rehabilitation group than that in the aspirin group (P<0.05). The photothermal pain tolerance threshold of the sham, aspirin, and aspirin+rehabilitation groups were significantly higher than those of the model group at 4, 6, and 8 weeks after operation (P<0.05). The inclination angles of the model, aspirin, and aspirin+rehabilitation groups were significantly lower than those of the sham group at 4, 6, and 8 weeks after operation, and the inclination angle of the aspirin+rehabilitation group was significantly higher than that of the model and aspirin groups (P<0.05). The sensory nerve regeneration distance in aspirin and aspirin+rehabilitation groups was higher than that in the sham and model groups (P<0.05). The expression of S100B protein in the aspirin and aspirin+rehabilitation groups was higher than that in the model group (P<0.05). Aspirin combined with rehabilitation training can promote the functional recovery of sciatic nerve injury, and the mechanism may be related to the increase of the expression of S100B protein in Schwann cells.
En este estudio se investigó el papel y el mecanismo que desempeña la aspirina combinada, con el entrenamiento de rehabilitación en la reparación de lesiones nerviosas y los cambios en los schwannocitos en ratas con lesiones en el nervio ciático. En total, 120 ratas SD macho sanas se dividieron aleatoriamente en cuatro grupos de 30 ratas en cada uno: simulación, modelo, aspirina y aspirina + rehabilitación. Se compararon el índice de función del nervio ciático (SFI), el umbral de tolerancia al dolor fototérmico y los resultados de la prueba del plano inclinado a las 4, 6 y 8 semanas después de la operación. Se analizó la distancia de regeneración del nervio sensorial y la expresión de la proteína S100B en los schwannocitos. En comparación con el grupo simulado, el SFI de los grupos modelo, aspirina y aspirina+rehabilitación fue significativamente menor a las 4, 6 y 8 semanas después de la operación. Sin embargo, los grupos de aspirina y aspirina + rehabilitación tuvieron un SFI significativamente más alto que el grupo modelo. El SFI a las 6 y 8 semanas después de la operación fue mayor en el grupo de aspirina + rehabilitación que en el grupo de aspirina (P<0,05). El umbral de tolerancia al dolor fototérmico de los grupos simulado, aspirina y aspirina+rehabilitación fue significativamente mayor que el del grupo modelo a las 4, 6 y 8 semanas después de la operación (P<0,05). Los ángulos de inclinación de los grupos modelo, aspirina y aspirina+rehabilitación fueron significativamente menores que los del grupo simulado a las 4, 6 y 8 semanas después de la operación, y el ángulo de inclinación del grupo aspirina+rehabilitación fue significativamente mayor que el de los grupos modelo y aspirina (P<0.05). La distancia de regeneración del nervio sensorial en los grupos de aspirina y aspirina+rehabilitación fue mayor que en los grupos simulado y modelo (P<0,05). La expresión de la proteína S100B en los grupos de aspirina y aspirina+rehabilitación fue mayor que en el grupo modelo (P<0,05). La aspirina combinada con el entrenamiento de rehabilitación puede promover la recuperación funcional de la lesión del nervio ciático, y el mecanismo puede estar relacionado con el aumento de la expresión de la proteína S100B en los schwannocitos.
Subject(s)
Animals , Rats , Sciatic Nerve/cytology , Exercise , Aspirin/therapeutic use , Sciatic Neuropathy/rehabilitation , Schwann Cells , Immunohistochemistry , Pain Threshold , Combined Modality Therapy , Sciatic Neuropathy/physiopathology , Disease Models, AnimalABSTRACT
Objective:To explore the effect of sciatic nerve derived exosomes(SN-EXO) on axon regeneration and functional recovery after peripheral nerve injury(PNI).Methods:From March 2021 to October 2022, the Department of Orthopedics of the First Affiliated Hospital of Zhengzhou University studied the effect of SN-EXO on the proliferation of Schwann cells(SCs) through EdU cell proliferation experiment. Twenty-one healthy male SD rats were randomly divided into 3 groups of sham operation, peripheral nerve injury(PNI) and SN-EXO treatment, with 7 rats in each group. The right sciatic nerves of rat models in sham group were exposed without injury. In the rat in PNI group and SN-EXO treatment group, PBS and SN-EXO were injected under the epineurium of right sciatic nerves following sciatic nerve crush. Sciatic nerve function index(SFI) was performed at 28 days after operation, and then sacrificed. Right sciatic nerves were removed for further exploration of nerve regeneration. The histopathological changes and axon arrangement of sciatic nerves were evaluated by HE staining. Regeneration efficiency of neurofilaments and SCs were obserred by NF200 and S100β double staining of sciatic nerve. The data obtained were statistically analyzed, and P<0.05 was statistically significant. Results:It was found that SN-EXO can significantly enhance the proliferation ability of SCs, with statistically significant difference( P<0.05). SFI in SN-EXO treatment group and PNI group were(-27.65±4.36) and(-57.33±7.49), respectively, and the difference was statistically significant( P<0.05). Axons in SN-EXO treatment group were arranged more closely and orderly than those in the PNI group at 28 days after operation, and there were less injury induced axon disintegration and vacuolation. Immunofluorescence assay indicated that NF200 and S100β fluorescence intensity in SN-EXO treatment group was significantly higher than that in the PNI group, and the difference was statistically significant( P<0.05). Conclusion:SN-EXO could enhance the proliferation of SCs to promote axon regeneration following peripheral nerve injury.
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Objective:To document any effect of environmental enrichment on nerve regeneration in a mouse model of sciatic nerve compression and explore its mechanism.Methods:A crushed sciatic nerve model was successfully established in 22 C57BL/6 mice, and they were then randomly divided into an intervention group and a control group. The mice of the intervention group were raised in a cage with an enriched environment, while those of the control group were kept in a standard cage. Two weeks later, both groups′ gait was analyzed and the compound muscle action potential (CMAP) of the sciatic nerve was measured. The proportion of myelinated sciatic nerve fibers was examined using toluidine blue staining, and the expression of myelin basic protein (MBP), growth associated protein-43 (GAP43) and p75 neurotrophin receptor (p75 NTR) was measured using immunofluorescence intensity. Results:①The latency of the CMAP [(1.05±0.04)ms] was significantly shortened in the intervention group compared with the control group and the amplitude was significantly higher. ②Gait analysis showed a significant increase in the average contact intensity, stride length and stride rate of the intervention group compared with the control group. However, the step axis angle of the intervention group was significantly smaller than in the control group on average. ③The stained nerve fibers in the intervention group were orderly and dense, and the average number of myelinated fibers was significantly greater than in the control group. ④Quantitative analysis of the immunofluorescence showed that the levels of MBP, GAP43 and p75 NTR in the sciatic nerves of the intervention group were, on average, significantly higher than in the control group. Conclusion:An enriched environmental can promote the regeneration and functional recovery of crushed sciatic nerves by promoting the proliferation and myelination of Schwann cells.
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OBJECTIVE To explore the mechanism of Taohong siwu decoction （THD） improving peripheral nerve injury induced by paclitaxel （PTX） in rats. METHODS The effects of THD （1 g/mL drug-containing serum） and PTX （0.1 μmol/L） alone or in combination on the proliferation rate of Schwann cells line RSC96 as well as the expressions of lysosomal-associated membrane protein-2 （LAMP2）， autophagy marker protein yeast Atg 6 homolog （Beclin1）， phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， and mammalian target of rapamycin （mTOR） were investigated， and then compared with autophagy promoter rapamycin and autophagy inhibitor 3-methyladenine （3-MA）. The effects of high-dose and low-dose THD on the expressions of myelin basic protein （MBP） and myelin protein zero （MPZ）， S100 calcium-binding protein （S100）， LAMP2， Beclin1， PI3K， Akt and mTOR were tested at the end of the experiment. RESULTS After treatment of THD+PTX， the proliferation rate of RSC96 cells was significantly higher than those treated with PTX alone. After treatment of THD+PTX or THD+ 3-MA， the protein expressions of LAMP2 and Beclin1 in RSC96 cells were significantly higher than those treated with PTX or 3- MA alone， while the protein expressions of PI3K， Akt and mTOR were significantly lower than those treated with PTX or 3-MA alone （P＜0.05）. Compared with model group， the protein expressions of MBP， MPZ， S100， LAMP2 and Beclin1 in sciatic nerve of rats were increased significantly in THD high-dose and low-dose groups， while the protein expressions of PI3K， Akt and mTOR were significantly decreased （P＜0.05）. CONCLUSIONS THD may activate Schwann cell autophagy by inhibiting the PI3K/Akt/ mTOR signaling pathway， thereby improving peripheral nerve injury caused by PTX.
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ObjectiveTo observe the effects of Hedysarum polysaccharides（HPS）on the signaling pathways of B-cell lymphoma 2 （Bcl-2）， cysteinyl aspartate-specific protease 3 （Caspase-3）， and Bcl-2-associated X protein （Bax） in Schwann cells（SCs）cultured in high glucose，and explore the possible mechanism of HPS against diabetic peripheral neuropathy（DPN）. MethodFour SD suckling mice aged 5-7 days were randomly divided into a normal group，a high-glucose group，an HPS + high-glucose group，and an α-lipoic acid（α-LA）+ high-glucose group. SCs were extracted from the sciatic nerve and cultured in a 37 ℃，5% CO2 incubator. After the cells reached 80% confluence，Cell Counting Kit-8（CCK-8）was used to screen the experimental concentrations suitable for high glucose，HPS， and α-LA interventions. Western blot and Real-time polymerase chain reaction （Real-time PCR）were used to detect the protein and mRNA expression of Bcl-2，Bax，and Caspase-3. The apoptosis rate of SCs was detected by flow cytometry using Annexin V-fluorescein isothiocyanate （FITC）/propidium iodide （PI）. ResultAs revealed by Western blot and real-time PCR，compared with the normal group，the high-glucose group showed reduced protein and mRNA expression of Bcl-2 and increased protein and mRNA expression of Bax and Caspase-3（P<0.01）. Compared with the high-glucose group，the HPS + high-glucose group and the α-LA + high-glucose group showed increased protein and mRNA expression of Bcl-2 and decreased protein and mRNA expression of Bax and Caspase-3（P<0.01）. As displayed by the results of flow cytometry using Annexin V/PI， compared with the normal group，the high-glucose group showed increased apoptosis rate；compared with the high-glucose group，the HPS + high-glucose group and the α-LA + high-glucose group showed reduced apoptosis rate（P<0.01）. ConclusionHPS can alleviate the apoptotic response of SCs，and its mechanism may be related to the inhibition of the activation of the Bcl-2/Caspase-3 signaling pathway.
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Enhancing remyelination after injury is of utmost importance for optimizing the recovery of nerve function. While the formation of myelin by Schwann cells (SCs) is critical for the function of the peripheral nervous system, the temporal dynamics and regulatory mechanisms that control the progress of the SC lineage through myelination require further elucidation. Here, using in vitro co-culture models, gene expression profiling of laser capture-microdissected SCs at various stages of myelination, and multilevel bioinformatic analysis, we demonstrated that SCs exhibit three distinct transcriptional characteristics during myelination: the immature, promyelinating, and myelinating states. We showed that suppressor interacting 3a (Sin3A) and 16 other transcription factors and chromatin regulators play important roles in the progress of myelination. Sin3A knockdown in the sciatic nerve or specifically in SCs reduced or delayed the myelination of regenerating axons in a rat crushed sciatic nerve model, while overexpression of Sin3A greatly promoted the remyelination of axons. Further, in vitro experiments revealed that Sin3A silencing inhibited SC migration and differentiation at the promyelination stage and promoted SC proliferation at the immature stage. In addition, SC differentiation and maturation may be regulated by the Sin3A/histone deacetylase2 (HDAC2) complex functionally cooperating with Sox10, as demonstrated by rescue assays. Together, these results complement the recent genome and proteome analyses of SCs during peripheral nerve myelin formation. The results also reveal a key role of Sin3A-dependent chromatin organization in promoting myelinogenic programs and SC differentiation to control peripheral myelination and repair. These findings may inform new treatments for enhancing remyelination and nerve regeneration.
Subject(s)
Animals , Rats , Axons , Chromatin/metabolism , Gene Expression Profiling , Myelin Sheath/metabolism , Nerve Regeneration/physiology , Schwann Cells/metabolism , Sciatic Nerve/injuriesABSTRACT
OBJECTIVES@#This study investigated the effects of different implant surface properties on the biological behavior of Schwann cells.@*METHODS@#Schwann cells (SCs) were cultured on three types of implant surfaces including smooth polished (SMO), sand-blasted, large grit, acid-etched (SLA), and chemically-modified SLA (modSLA). At different time points, the morphology and adhesion of SCs on the implant surfaces were observed by scanning electron microscope. Cell proliferation activity was detected by MTT method. The expression levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were detected by enzyme-linked immunosorbent assay. Changes in the mRNA levels of NGF and BDNF were detected by real-time fluorescent quantitative polymerase chain reaction (PCR).@*RESULTS@#SCs adhered, stretched, and proliferated well on the three types of implant surfaces. On the 3rd, 5th, and 7th days, the OD values of the SMO group were higher than those of the SLA group and the modSLA group, and the difference was statistically significant (@*CONCLUSIONS@#Different implant surface properties have different effects on the biological behavior of SCs. Proliferation of SCs is significantly promoted by smooth surface, while secretion and gene expression of neurotrophic factors are significantly promoted by modSLA surface at early stage.
Subject(s)
Dental Implants , Schwann Cells , Surface Properties , TitaniumABSTRACT
Peripheral nerve injury is common in clinical practice. Today, outcomes of peripheral nerve repair are still not satisfactory. Recently, a large number of studies have shown that exosomes and their bioactive substances can repair peripheral nerve injury by mediating axonal regeneration, activating Schwann cells, regulating inflammation and other pathways to restore nerve function. This review aims to summarise and prospect the role and application of exosomes in peripheral nerve repair from the perspective of Schwann cells, mesenchymal stem cells and macrophages derived exosomes and their bioactive substances.
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BACKGROUND: Non-coding RNA is widely distributed in the nervous system in vivo and a significant change in the expression of non-coding RNA has been observed in a neural injury model. This suggests that non-coding RNA may serve as a potential target for resolving the challenges of peripheral nerve repair. OBJECTIVE: To summarize the mechanisms of microRNA, circular RNA and long non-coding RNA in the process of repair after peripheral nerve injury with the attempt to determine the possible treatment of peripheral nerve injury. METHODS: The first author retrieved the relevant literatures in CNKI and PubMed databases published from January 2001 to April 2019. The key words were “non-coding RNA, miRNA, circRNA, IncRNA, peripheral nerve injury” in Chinese and English, respectively. Forty-three literatures were included in accordance with the exclusion and inclusion criteria. RESULTS AND CONCLUSION: (1) MicroRNAs can act on certain signal pathways, regulate the apoptosis, growth, proliferation and differentiation of Schwann cells and participate in the repair of peripheral nerve injury. (2) Circular RNAs act as microRNA sponges to competitively inhibit the transcription in microRNA, and exert corresponding biological functions. (3) A large amount of long non-coding RNAs are expressed after peripheral nerve injury, and play a key role in the peripheral nerve regeneration.
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OBJECTIVE: Schwann cells can promote the regeneration of damaged peripheral nerves and serve as seed cells in the engineering repair of peripheral nerve tissue. The effective culture and purification of Schwann cells are the basis of clinical treatment for peripheral nerve injury. This paper summarized the literature of culture of Schwann cells in vitro in recent ten years and made a descriptive review on the research progress of Schwann cell culture and purification, in order to provide references for culture of Schwann cells in vitro. METHODS: Literature related to Schwann cell isolation, culture and purification was retrieved from PubMed, Web of Science, Medline databases, CNKI, Wanfang, VIP and other databases. The keywords included “Schwann cells; isolation; culture; purification”. All the papers obtained from the search were read, analyzed and judged, and finally 62 papers meeting the standards were included. RESULTS: The classical methods of Schwann cell culture include tissue block culture and enzyme digestion. Purification methods include mitosis resistance, immune selection, specific adhesion, pre degeneration, cold jet, differential adherent, laminin package, low serum, stimulating factor, fluorescence activated cell sorting or magnetic activated cell sorting, immunopanning, and extracorporeal shock wave treatment. CONCLUSION: Schwann cells can be cultured and purified in various ways in vitro, and each method has its advantages and disadvantages. How to obtain high-purity Schwann cells quickly and efficiently is still a challenge. Multiple methods can be combined for purification and affective factors can be controlled for Schwann cell proliferation as much as possible.
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@#Peripheral nerve injury (PNI) is a common disease in the oral cavity that can easily lead to loss of function and abnormal appearance. The application of dental pulp stem cells (DPSCs) combined with tissue engineering in the repair of PNI is a research hotspot. DPSCs have the advantages of abundant sources, simple extraction, low immunogenicity and a high proliferation rate in vitro. They can differentiate into Schwann cells (SCs). SCs can induce autophagy and secrete key neurotrophic factors, such as nerve growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor and glial cell-derived neurotrophic factor. SCs are beneficial for the repair of nerve injury. DPSCs in different periods have differences in immune regulation, anti-inflammatory effects, expression of neural markers, angiogenesis and so on, which provide more diversified choices for nerve repair. At present, the introduction of tissue engineering provides a more controllable and improved microenvironment for DPSCs, which is conducive to the application and development of DPSCs in regenerative medicine and tissue engineering. However, there are still many problems to be solved, such as the selection of stem cells, functional link recovery, uncontrollable direction of axon regeneration, regulation of the peripheral nervous system and mechanism of repair.
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This study was to investigate the protective effect of paeoniflorin (PF) on hydrogen peroxide-induced injury. Firstly, "SMILES" of PF was searched in Pubchem and further was used for reverse molecular docking in Swiss Target Prediction database to obtain potential targets. Injury-related molecules were obtained from GeenCards database, and the predicted targets of PF for injury treatment were selected by Wayne diagram. For mechanism analysis, the protein-protein interactions were constructed by String, and the KEGG analysis was conducted in Webgestalt. Then, cell viability and cytotoxicity assay were established by CCK8 assay. Also, the experimental cells were allocated to control, model (200 μmol·L
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PURPOSE@#Wallerian degeneration (WD) is an antegrade degenerative process distal to peripheral nerve injury. Numerous genes are differentially regulated in response to the process. However, the underlying mechanism is unclear, especially the early response. We aimed at investigating the effects of sciatic nerve injury on WD via CLDN 14/15 interactions in vivo and in vitro.@*METHODS@#Using the methods of molecular biology and bioinformatics analysis, we investigated the molecular mechanism by which claudin 14/15 participate in WD. Our previous study showed that claudins 14 and 15 trigger the early signal flow and pathway in damaged sciatic nerves. Here, we report the effects of the interaction between claudin 14 and claudin 15 on nerve degeneration and regeneration during early WD.@*RESULTS@#It was found that claudin 14/15 were upregulated in the sciatic nerve in WD. Claudin 14/15 promoted Schwann cell proliferation, migration and anti-apoptosis in vitro. PKCα, NT3, NF2, and bFGF were significantly upregulated in transfected Schwann cells. Moreover, the expression levels of the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK signaling pathways were also significantly altered.@*CONCLUSION@#Claudin 14/15 affect Schwann cell proliferation, migration, and anti-apoptosis via the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK pathways in vitro and in vivo. The results of this study may help elucidate the molecular mechanisms of the tight junction signaling pathway underlying peripheral nerve degeneration.
Subject(s)
Animals , Rats , Claudins , Nerve Regeneration , Peripheral Nerve Injuries , Schwann Cells/pathology , Sciatic Nerve , Wallerian Degeneration/pathologyABSTRACT
Mucosal Schwann cell hamartoma (MSCH) is a rare benign neurogenic tumor characterized by pure S100p positive spindle cell proliferation. Most cases occur in the distal colon. Involvement of the gall bladder is exceedingly rare. There have been no reports of recurrence or a syndromic association with MSCH. Herein, we describe a case of MSCH of the gallbladder in a 55-year-old female patient with prior history of gastrointestinal neurofibromas who presented with abdominal pain. MR imaging revealed choledocholithiasis, gallbladder thickening, and marked biliary and pancreatic ductal dilation. The patient subsequently underwent cholecystectomy with choledochoduodenostomy. Histologic evaluation of the gallbladder showed diffuse expansion of the mucosa with S100p positive cells with spindly nuclei and indistinct cytoplasmic borders and diagnosis of MSCH of the gallbladder was rendered.
Subject(s)
Humans , Female , Middle Aged , Schwann Cells/pathology , Gallbladder Neoplasms/pathology , Hamartoma/pathology , Neurofibroma/pathology , NeuromaABSTRACT
Abstract Objective: To review the imaging features of granular cell tumors of the breast (on mammography, ultrasound, and magnetic resonance imaging), establishing a pathological correlation, in order to familiarize radiologists with this entity and make them aware of the differential diagnoses, other than malignancy, of lesions with spiculated margins. Materials and Methods: We reviewed the medical records (from a clinical-pathology database and picture archiving and communication system) of five patients with a pathologically confirmed diagnosis of granular cell tumor of the breast, treated at the Portuguese Oncology Institute of Lisbon, in the city of Lisbon, Portugal, between January 2012 and December 2018. Results: All five tumors exhibited imaging features highly suggestive of malignancy (BI-RADS 5 lesions), namely spiculated margins, significant depth, and posterior acoustic shadowing (on ultrasound). One tumor showed a kinetic curve indicative of washout on magnetic resonance imaging, two were adherent to the pectoralis muscle, and one was accompanied by skin retraction. Pathology provided the definitive diagnosis in all cases. Conclusion: Granular cell tumors of the breast pose a diagnostic challenge because they can present with clinical and imaging features mimicking malignancy, and the diagnosis is therefore provided by pathology. Radiologists should be familiarized with this entity, so they can be aware of the fact that breast lesions with spiculated margins can be indicative of diagnoses other than malignancy.
Resumo Objetivo: Rever as características de imagem dos tumores de células granulares da mama (mamográficas, ultrassonográficas e de ressonância magnética), estabelecendo uma correlação anatomopatológica, no intuito de proporcionar aos radiologistas uma familiarização com esta entidade e alertar para outros diagnósticos diferenciais de lesões espiculadas além das malignas. Materiais e Métodos: Consulta dos processos clínicos (base de dados clínica, anatomopatológica e sistema de comunicação e arquivamento de imagens) de doentes seguidos no Instituto Português de Oncologia de Lisboa, com diagnóstico anatomopatológico confirmado de tumor de células granulares da mama, de janeiro de 2012 a dezembro de 2018. Resultados: Todos os tumores exibiram características de imagem altamente sugestivas de malignidade (BI-RADS 5), nomeadamente espiculações, crescimento em profundidade e atenuação posterior (ultrassonografia), um mostrou um perfil cinético com washout na ressonância magnética, dois estavam aderentes ao músculo peitoral e um associava retração cutânea. O diagnóstico definitivo foi anatomopatológico. Conclusão: Os tumores de células granulares da mama constituem um desafio diagnóstico, pois podem apresentar características clínicas e de imagem que mimetizam malignidade, pelo que o diagnóstico é anatomopatológico. Os radiologistas devem estar familiarizados com esta entidade de forma a considerá-la nos diagnósticos diferenciais de lesões espiculadas, além das lesões malignas.
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Os cimentos dentários e ortopédicos são utilizados amplamente em diversas aplicações clínicas. Novos cimentos vêm sendo propostos visando à preservação ou regeneração tecidual. Contudo, pouco se conhece sobre o papel desses biomateriais na regeneração nervosa. As células mais comumente envolvidas na regeneração nervosa são as células de Schwann (SCs) sendo sua principal função o suporte aos axônios através da liberação de fatores de crescimento e isolamento axonal através da formação da bainha de mielina. Como estratégia da presente pesquisa, foi estudado um cimento à base da quitosana com adição de substâncias que podem atuar sinergicamente na resposta celular nervosa, tais como, as nanopartículas (NPs) de hidroxiapatita e o óxido de zinco, visto que têm propriedades bioativas e biocondutoras, além de promoverem a condução de prolongamento axonal. A doxiciclina (Dox) foi acrescida como antimicrobiano, potente inibidora de metaloproteinases (MMPs) e estimuladora da diferenciação celular no processo de regeneração tecidual. Assim, as propriedades físico-químicas e biológicas do cimento de nano-hidroxiapatita, quitosana, óxido de zinco e doxiciclina foram avaliadas, bem como a capacidade de promover um ambiente favorável para as células nervosas periféricas. Os cimentos foram caracterizados físico-químicamente mediante a determinação do pH, tempo de presa e solubilidade, lixiviação de íons cálcio, liberação controlada de doxiciclina, difração de Raios X, Termogravimetria (TG), espectroscopia Raman, molhabibidade, e testes de atividade biológica, para assim também serem avaliados em contato com células nervosas de Schwann (HS-Sch-2). O cimento apresentou pH neutro (7,0), tempo de presa de 5,7 ± 0,22 minutos, solubilidade menor que 3%, lixiviação de cálcio de 8,14 ± 0,71 mg L-1 após 14 dias, estabilidade térmica e a análise espectroscópica ratificou a presença e diferenciação das estruturas químicas dos componentes do cimento coerentemente com as imagens das análises microscópicas. Além disso, o cimento se mostrou hidrofílico, teve efeito hemolítico baixo (17%), obteve alta citocompabilidade celular em fibroblastos ATCC 3T3 (72%) e ação antimicrobiana. O cimento aumentou significativamente o crescimento das células de Schwann, 48,6% a mais do que o grupo controle (p≤0.05), e maior capacidade metabólica na análise mitótica quando em contato com este material (33%). Pode-se concluir que o cimento proposto à base de quitosana contendo hidroxiapatita e óxido de zinco nanoparticulados com adição de doxiciclina obteve efeito bioativo em células de Schwann promovendo, assim, o crescimento e a atividade mitótica celular, sendo então um biomaterial promissor para estudos de remielinização de nervos periféricos e regeneração nervosa in vivo.
Dental and orthopedic cements are used widely in several clinical applications. New cements have been proposed aimed the tissue preservation or regeneration. Nevertheless, nerve regeneration is not well known. The cells most commonly used in nerve regeneration are Schwann cells (SCs) which represent glial cells in the peripheral nervous system, their main function being supporting axons by releasing growth factors and axonal isolation through the formation of the myelin sheath. As a strategy of this research, chitosan-based cement was studied with the addition of substances that can act synergistically in the nervous cell response, such as the hydroxyapatite and zinc oxide nanoparticles (NPs), since they have bioactive and bioconductive properties; in addition to furthermore, they promote the conduction of axonal prolongation. Doxycycline (Dox) was added as an antimicrobial, a potent inhibitor of MMPs, a stimulator of cell differentiation in the tissue regeneration process. Thus, the physical-chemical and biological properties of nanohydroxyapatite, chitosan, zinc oxide and doxycycline cements were evaluated, as well as the ability to promote a favorable environment for peripheral nerve cells. Blocks of cements were characterized physically and chemically by determining pH, setting time and solubility, calcium ions leaching, controlled release of doxycycline, X-ray diffraction, Thermogravimetry (TG), Raman spectroscopy, wetness, and biological activity tests, so they can also be evaluated in contact with Schwann nerve cells (HS-Sch-2). The cement showed neutral pH (7.0), setting time of 5.7 ± 0.22 minutes, solubility less than 3%, calcium leaching of 8.14 ± 0.71 mg L-1 after 14 days, stability thermal and spectroscopic analysis confirmed the presence and differentiation of the chemical structures of the cement components coherently with the images of the microscopic analysis. In addition, the cement was shown to be hydrophilic, had a low hemolytic effect (17%), and obtained high cell cytocompatibility in ATCC 3T3 fibroblasts (72%) and antimicrobial action. The cement significantly increased the growth of Schwann cells, 48.6% more than the control group (p≤0.05), and greater metabolic capacity in the mitotic analysis when in contact with this material (33%). It can be concluded that the proposed chitosan-based cement containing hydroxyapatite and zinc oxide nanoparticulated with the addition of doxycycline has a bioactive effect in Schwann cells, thus promoting cell growth and mitotic activity, thus being a promising biomaterial for studies of remyelinization of peripheral nerves and nerve regeneration in vivo.
Subject(s)
Schwann Cells , Stimulation, Chemical , Dental Cements , Nerve Regeneration , Doxycycline , Durapatite , ChitosanABSTRACT
BACKGROUND Although Mycobacterium leprae (ML) is well characterised as the causative agent of leprosy, the pathophysiological mechanisms underlying peripheral nerve damage still need further understanding. In vitro and in vivo studies have yielded insights into molecular mechanisms of ML interaction with Schwann cells (SC), indicating the regulation of genes and proteins crucial to neural plasticity. OBJECTIVES We aimed to investigate the effect of ML on neurotrophins expression in human SC (hSC) and mice sciatic nerves to better understand their role in leprosy neuropathy, and aiming to contribute to future therapeutic approaches. METHODS We evaluated mRNA and protein expression of BDNF, NGF, NT-3, NT-4 in hSC from amputation nerve fragments, as well as in athymic nude mice, infected by ML for eight months. FINDINGS and MAIN CONCLUSIONS Our in vitro results showed a trend to decline in NGF and BDNF mRNA in ML-treated hSC, compared to controls. The immunodetection of BDNF and NT-4 was significantly downregulated in ML-treated hSC. Conversely, ML-infected mice demonstrated upregulation of NT-3, compared to non-infected animals. Our findings indicate that ML may be involved in neurotrophins regulation, suggesting that a pathogen-related imbalance of these growth factors may have a role in the neural impairment of leprosy.
Subject(s)
Humans , Animals , Mice , Schwann Cells/metabolism , Sciatic Nerve/metabolism , Mycobacterium leprae , Nerve Growth Factors/metabolism , Mice, NudeABSTRACT
OBJECTIVE@#To investigate the therapeutic and synergistic effects of QHC (combination of quercetin (Q), hirudin (H) and cinnamaldehyd (C)) on Schwann cell differentiation and myelination against high glucose (HG) induced injury.@*METHODS@#Primary-culture Schwann cells exposed to HG (50 mmol/L) for 72 h and Schwann cell-dorsal root ganglion (DRG) neuron cocultures exposed to HG (50 mmol/L) for 7 days were employed as in vitro model of diabetic neuropathy. The cells were randomly divided into 10 groups: control (CON, 25 mmol/L glucose), HG (50 mmol/L glucose), HG plus 10 μmol/L quercetin (Q), HG plus 0.04 IU/mL hirudin (H), HG plus 100 nmol/L cinnamaldehyd (C), HG plus 10 μmol/L quercetin and 0.04 IU/mL hirudin (QH), HG plus 10 μmol/L quercetin and 50 nmol/L cinnamaldehyd (QC), HG plus 0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd (HC), HG plus 10 μmol/L quercetin, 0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd (QHC) or 10 μmol/L U0126. Cell differentiation was evaluated by periaxin immunofluorescence staining. The protein expression levels of myelin protein zero (P0), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), extracellular signal-regulated kinase (ERK), p-ERK, p-c-Jun, c-Jun, notch intracellular domain (NICD) and the mRNA expression levels of P0, MBP, MAG, Krox-20, Notch1 and Jagged1 were detected by Western blotting and real-time quantitative PCR analysis. The secretion of ciliary neurotrophic factor (CNTF) was determined by enzyme-linked immunosorbent assay (ELISA). The number and length of the myelin segments were evaluated by MBP immunofluorescence staining. The expression and the location of p-ERK in cocultures were detected by MAG and p-ERK immunofluorescence double staining.@*RESULTS@#Co-treatment with Q, C, H and their combination promoted Schwann cell differentiation, increased CNTF secretion, up-regulated the protein and mRNA expressions of myelin, and increased the number and length of the myelin segments (P<0.01 or P<0.05). In particular, the combination therapy of Q, H and C was superior to the respective monotherapy (P<0.01). Combination therapy of QHC exhibited higher inhibitory activities for ERK signaling related molecules than each monomer or the combination of the two monomers (P<0.01).@*CONCLUSION@#QHC combination yielded synergy in promoting Schwann cell differentiation and myelination and the protective effect may involve in the inhibition of ERK signaling pathway, providing scientific evidence for better understanding of combination of Q, H and C in clinical applications.
ABSTRACT
BACKGROUND: Recent studies have shown that the occurrence of Wallerian degeneration is closely related to autophagy in Schwann cells. The regulation of autophagy in Schwann cells can significantly affect the occurrence and development of Wallerian degeneration, subsequently altering axon regeneration and myelination. OBJECTIVE: To clarify whether sciatic nerve allograft can achieve higher efficiency when the cell autophagy is inhibited by 3-methyladenine. METHODS: We harvested 16 sciatic nerve segments from 8 female C57BL/6J mice that were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. in China. All the segments were equally divided into experimental and control groups and cultured in 3-methyladenine culture medium and normal culture medium for 72 hours, respectively. Another 16 female C57BL/6J mice were taken to make animal models of left sciatic nerve defects. After modeling, the sciatic nerve segments were grafted to repair sciatic nerve defect through microsurgery: 3-methyladenine-treated nerve segments in the experimental group and normally treated nerve segments in the control group. Sciatic nerve index in each mouse was recorded at 2, 4, 6, and 8 weeks after modeling. At 8 weeks after modeling, the regenerated nerve segments were histologically analyzed using hematoxylin-eosin staining, immunofluorescent staining, toluidine blue staining, and transmission electron. The animal experiment was approved by the Animal Ethics Committee of Peking Union Medical College. RESULTS AND CONCLUSION: There were no differences in the sciatic nerve index between the two groups (P > 0.05) except at 8 weeks after modeling (P < 0.05). Hematoxylin-eosin staining revealed an intact nerve structure in the experimental group but a large area of voids in the control group. Immunofluorescent staining indicated that there were nerve tracts with more complete structures in the experimental group than the control group. Toluidine blue staining revealed some myelinated and unmyelinated nerve fibers regenerated in the experimental group and only a few of myelinated nerve fibers and unmyelinated axons newly formed in the control group. Under the transmission electron microscope, myelin sheath thickness and myelinated fiber diameter were significantly higher in the experimental group than the control group (P < 0.05). Therefore, 3-methyladenine-treated nerve allografts could inhibit autophagy in Schwann cells, maintain the myelin sheath structure of the allograft, and promote axonal regeneration and functional recovery.
ABSTRACT
BACKGROUND: The clinical effect of spinal cord injury is usually unfavorable due to the lack of axon regeneration and the formation of glial scar. Schwann cells, as the support cells for nerve regeneration, have poor migration ability in the central nervous system with abundant astrocytes, which limit its effect on axon regeneration. OBJECTIVE: To explore the effect on the migration of Schwann cells containing superparamagnetic nanoparticles loaded with chondroitinase ABC (ChABC) in the region of astrocytes in the external magnetic field. METHODS: Schwann cells and astrocytes were extracted from sciatic nerves and brachial plexus and cerebral cortex of Sprague-Dawley rats of postnatal day 1 to 3. Cell purity was identified by immunofluorescence staining. The toxicity of superparamagnetic nanoparticles (PEI-SPIONs) to Schwann cells was analyzed by live/dead cell staining. Schwann cells were transfected with PEI-SPIONS in an external magnetic field of 1.4Td for 2 days. The optimal transfection concentration of PEI-SPIONS used was 2 mg/L and the optimal mass ratio of PEI-SPIONS to ChABC was 1:4. Cell migration test was used to evaluate the migration ability of not-treated Schwann cells, PEI-SPIONs/ ChABC transfected Schwann cells, and PEI-SPIONs/ChABC transfected Schwann cells in an external magnetic field. RESULTS AND CONCLUSION: The purity of Schwann cells and astrocytes reached to (91.7±1.2)% and (93.3±2.2)%, respectively. Compared with the Schwann cells group, the number of PEI-SPIONs/ChABC-transfected Schwann cells that entered the region of astrocytes was significantly increased (P < 0.05). Under the external magnetic field, the number of PEI-SPIONs/ChABC-transfected Schwann cells that entered the region of astrocytes and the cell migration distance were significantly increased as compared with the Schwann cells group (P < 0.005). In summary, PEI-SPIONs/ChABC transfection can enhance the ability of Schwann cells to break the glial scar, and increase the fusion of astrocytes. Under the guidance of external magnetic field, the migration ability of Schwann cells is significantly elevated. This method may be a new strategy to promote nerve regeneration after spinal cord injury.