RESUMO
BACKGROUND: Neuroinflammation is a characteristic pathological change of acute neurological deficit and chronic traumatic encephalopathy (CTE) after traumatic brain injury (TBI). Microglia are the key cell involved in neuroinflammation and neuronal injury. The type of microglia polarization determines the direction of neuroinflammation. MiR-21-5p elevated in neurons and microglia after TBI in our previous research. In this study, we explore the influence of miR-21-5p for neuroinflammation by regulating microglia polarization. METHODS: In this study, PC12 and BV2 used to instead of neuron and microglia respectively. The co-cultured transwell system used to simulate interaction of PC12 and BV2 cells in vivo environment. RESULTS: We found that PC12-derived exosomes with containing miR-21-5p were phagocytosed by microglia and induced microglia polarization, meanwhile, the expression of miR-21-5p was increased in M1 microglia cells. Polarization of M1 microglia aggravated the release of neuroinflammation factors, inhibited the neurite outgrowth, increased accumulation of P-tau and promoted the apoptosis of PC12 cells, which formed a model of cyclic cumulative damage. Simultaneously, we also got similar results in vivo experiments. CONCLUSIONS: PC12-derived exosomes with containing miR-21-5p is the essential of this cyclic cumulative damage model. Therefore, regulating the expression of miR-21-5p or the secretion of exosomes may be an important novel strategy for the treatment of neuroinflammation after TBI.
Assuntos
Diferenciação Celular , Exossomos/genética , MicroRNAs/genética , Microglia/citologia , Neurônios/citologia , Animais , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Técnicas de Cocultura , Exossomos/metabolismo , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos , Células PC12 , RatosRESUMO
Neuronal inflammation is the characteristic pathologic change of acute neurologic impairment and chronic traumatic encephalopathy after traumatic brain injury (TBI). Inhibiting the excessive inflammatory response is essential for improving the neurologic outcome. To clarify the regulatory mechanism of microglial exosomes on neuronal inflammation in TBI, we focused on studying the impact of microglial exosomal miRNAs on injured neurons in this research. We used a repetitive (r)TBI mouse model and harvested the injured brain extracts from the acute to the chronic phase of TBI to treat cultured BV2 microglia in vitro The microglial exosomes were collected for miRNA microarray analysis, which showed that the expression level of miR-124-3p increased most apparently in the miRNAs. We found that miR-124-3p promoted the anti-inflamed M2 polarization in microglia, and microglial exosomal miR-124-3p inhibited neuronal inflammation in scratch-injured neurons. Further, the mammalian target of rapamycin (mTOR) signaling was implicated as being involved in the regulation of miR-124-3p by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Using the mTOR activator MHY1485 we confirmed that the inhibitory effect of exosomal miR-124-3p on neuronal inflammation was exerted by suppressing the activity of mTOR signaling. PDE4B was predicted to be the target gene of miR-124-3p by pathway analysis. We proved that it was directly targeted by miR-124-3p with a luciferase reporter assay. Using a PDE4B overexpressed lentivirus transfection system, we suggested that miR-124-3p suppressed the activity of mTOR signaling mainly through inhibiting the expression of PDE4B. In addition, exosomal miR-124-3p promoted neurite outgrowth after scratch injury, characterized by an increase on the number of neurite branches and total neurite length, and a decreased expression on RhoA and neurodegenerative proteins [Aß-peptide and p-Tau]. It also improved the neurologic outcome and inhibited neuroinflammation in mice with rTBI. Taken together, increased miR-124-3p in microglial exosomes after TBI can inhibit neuronal inflammation and contribute to neurite outgrowth via their transfer into neurons. miR-124-3p exerted these effects by targeting PDE4B, thus inhibiting the activity of mTOR signaling. Therefore, miR-124-3p could be a promising therapeutic target for interventions of neuronal inflammation after TBI. miRNAs manipulated microglial exosomes may provide a novel therapy for TBI and other neurologic diseases.-Huang, S., Ge, X., Yu, J., Han, Z., Yin, Z., Li, Y., Chen, F., Wang, H., Zhang, J., Lei, P. Increased miR-124-3p in microglial exosomes following traumatic brain injury inhibits neuronal inflammation and contributes to neurite outgrowth via their transfer into neurons.
Assuntos
Lesões Encefálicas Traumáticas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Microglia/metabolismo , Animais , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Modelos Animais de Doenças , Exossomos/genética , Exossomos/metabolismo , Redes Reguladoras de Genes , Inflamação/genética , Inflamação/metabolismo , Inflamação/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/classificação , Microglia/patologia , Crescimento Neuronal/genética , Crescimento Neuronal/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND/AIMS: High concentration of bile acids (BAs) induces hydrophobicity-dependent vasorelaxtant effects with hydrophobic BAs showing greater responses than hydrophilic BAs, of which the underlying mechanisms are still unclear. Caveolae are invaginations on membranes of endothelial cells (ECs) entraping endothelial nitric oxide synthase (eNOS) to prevent its activation, which plays a critical role in regulation of vascular function. The purpose of the present study was to investigate the role of caveolae in vasorelaxant effects of BAs. METHODS: Chenodeoxycholic acid (CDCA) and cholic acid (CA) were used to represent hydrophobic and hydrophilic BA, respectively. Vascular responses of abdominal aorta were measured by isometric force recording. Morphology of caveolae was examined by transmission electron microscopy. Protein expression of total eNOS (t-eNOS) or phosphorylated eNOS (p-eNOS) was determined by Western blot. Nitric oxide (NO) content was observed by fluorometric assay. RESULTS: We demonstrated that CDCA as well as Methyl-ß-cyclodextrin (MCD), a commonly used reagent for cholesterol depletion, reduced potassium chloride (KCl)- or phenylephrine (PE)-elicited vasoconstriction (P < 0.05), and enhanced acetylcholine (Ach)-elicited vasodilatation (P < 0.05) in endothelium-intact abdominal aorta but not in endothelium-denuded or CA-treated vessels. CDCA and MCD, but not CA significantly disrupted caveolae structure on ECs of abdominal aorta which was recovered by cholesterol incubation (P < 0.05). Protein expression of t-eNOS was significantly decreased (P < 0.05), and that of p-eNOS together with NO content was significantly increased in CDCA- and MCD- but not CA-treated vessels (P < 0.05) as compared with vehicle. The effect was reversed by either endothelium-denudation or cholesterol replenishment. Moreover, with cholesterol incubation, no significant differences were found in vascular responses among CDCA-, CA- or vehicle-treated vessels. CONCLUSION: These results indicate that CDCA diminishes caveolae on ECs of abdominal aorta promoting eNOS phosphorylation and NO production which contributes to its vasorelaxtant effect.
Assuntos
Aorta/efeitos dos fármacos , Cavéolas/efeitos dos fármacos , Ácido Quenodesoxicólico/farmacologia , Endotélio Vascular/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Aorta/fisiologia , Cavéolas/metabolismo , Cavéolas/ultraestrutura , Ácido Cólico/farmacologia , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Masculino , Óxido Nítrico/análise , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/análise , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacosRESUMO
Weightlessness induces the functional remodelling of arteries, but the changes to angiotensin II (Ang II)-elicited vasoconstriction and the underlying mechanism have never been reported. Caveolae are invaginations of the cell membrane crucial for the contraction of vascular smooth muscle cells, so we investigated the adaptation of Ang II-elicited vasoconstriction to simulated weightlessness and the role of caveolae in it. The 4 week hindlimb unweighted (HU) rat was used to simulate the effects of weightlessness. Ang II-elicited vasoconstriction was measured by isometric force recording. The morphology of caveolae was examined by transmission electron microscope. The binding of the angiotensin II type 1 receptor (AT1 ) and caveolin-1 (cav-1) was examined by coimmunoprecipitation and Western blot. We found that the maximal developing force (E(max)) of Ang II-elicited vasoconstriction was decreased in abdominal aorta by 30.6%, unchanged in thoracic aorta and increased in carotid artery by 17.9% after HU, while EC50 of the response was increased in all three arteries (P < 0.05). AT1 desensitization upon activation was significantly reduced by HU in all three arteries, as was the number of caveolae (P < 0.05). Furthermore, Ang II promoted the binding of AT1 and cav-1 significantly in control but not HU arteries. Both the number of caveolae and the binding of AT1 and cav-1 in HU arteries were restored by cholesterol pretreatment which also reinstated the change in EC50 as well as the level of AT1 desensitization. These results indicate that modified caveolae in vascular smooth muscle cells could interfere with the binding of AT1 and cav-1 mediating the adaptation of Ang II-elicited vasoconstriction to HU.
Assuntos
Angiotensina II/farmacologia , Aorta Abdominal/fisiologia , Aorta Torácica/fisiologia , Artérias Carótidas/fisiologia , Cavéolas/fisiologia , Vasoconstrição/efeitos dos fármacos , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Torácica/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Caveolina 1/metabolismo , Colesterol/farmacologia , Membro Posterior , Elevação dos Membros Posteriores/fisiologia , Masculino , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismo , Vasoconstrição/fisiologia , Ausência de PesoRESUMO
Here we report the assembly of multilayered DNA nanocages. The layers can be separated in response to a chemical cue, ATP. This is an effort to increase the structural complexity of DNA nanocages. The structures have been characterized by native polyacrylamide gel electrophoresis, atomic force microscopy, and cryogenic electron microscopy. We envision that the layer-by-layer assembly strategy used in this study can be easily applied to other DNA nanocages to form Russian-doll-like semisolid structures, while the chemically activated layer separation makes a contribution to the development of "smart" DNA nanocages.
Assuntos
DNA/química , Nanotecnologia/métodos , Trifosfato de Adenosina/química , Modelos Moleculares , Conformação MolecularRESUMO
Microgravity-induced vascular remodelling may play an important role in post-spaceflight orthostatic intolerance. In this study, we aimed to investigate the effects of simulated microgravity on monocyte adhesion to aortic endothelium in hindlimb unweighted rats and to elucidate the underlying mechanisms associated with this event. Sprague-Dawley rats were subjected to 4-week hindlimb unweighting to simulate microgravity. The recruitment of monocytes to the abdominal aorta was investigated by en face immunofluorescence staining and monocyte binding assays. The expression of the adhesion molecules E-selectin and vascular cell adhesion molecule-1 as well as the cytokine monocyte chemoattractant protein (MCP)-1 was evaluated by immunohistochemical staining, western blot, and quantitative reverse transcription polymerase chain reaction analyses. Additionally, nuclear factor-κB (NF-κB) activation and the messenger RNA expression levels of E-selectin, vascular cell adhesion molecule-1, and MCP-1 were assessed with the administration of an NF-κB inhibitor, pyrrolidine dithiocarbamate. Results showed that simulated microgravity significantly increased monocyte recruitment to the aortic endothelium, protein expression of E-selectin and MCP-1, and NF-κB activation in the abdominal aorta of rats. Pyrrolidine dithiocarbamate treatment not only significantly inhibited NF-κB activity but also reduced the messenger RNA levels of E-selectin, vascular cell adhesion molecule-1, and MCP-1 as well as monocyte recruitment in the abdominal aorta of hindlimb unweighted rats. These results suggest that simulated microgravity increases monocyte adhesion to rat aortic endothelium via the NF-κB-mediated expression of the adhesion molecule E-selectin and the cytokine MCP-1. Therefore, an NF-κB-mediated inflammatory response may be one of the cellular mechanisms responsible for arterial remodelling during exposure to microgravity.
Assuntos
Aorta Abdominal/citologia , Endotélio Vascular/citologia , Monócitos/citologia , NF-kappa B/metabolismo , Simulação de Ausência de Peso , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quimiocina CCL2/genética , Selectina E/genética , Endotélio Vascular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Monócitos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Pirrolidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tiocarbamatos/farmacologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Nanotubes with different sizes can be readily assembled from simple DNA nanomotifs, which consist of just a few unique DNA sequences. Such structurally well-defi ned DNA-nanotubes will have great potential in many technological applications ranging from drug delivery, to determination of biomacromolecular 3D structures, to nanoplasmonic devices.
Assuntos
DNA/química , Nanotubos/química , Microscopia Crioeletrônica , Eletroforese em Gel de Poliacrilamida , Nanotubos/ultraestrutura , Conformação de Ácido Nucleico , Motivos de NucleotídeosRESUMO
Post-spaceflight orthostatic intolerance is one of the most important adverse effects after exposure to space microgravity, and there are still no effective countermeasures. It has been considered that arterial remodeling may play an important role in the occurrence of post-spaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. In this study, we investigated whether an inflammatory response exists in the common carotid artery of rats exposed to simulated microgravity. For this, Sprague-Dawley rats were subjected to 4 weeks of hindlimb unweighting to simulate microgravity. The expression levels of the adhesion molecules E-selectin and vascular cell adhesion molecule-1 (VCAM-1), and the cytokine monocyte chemoattractant protein-1 (MCP-1) in the common carotid artery of simulated microgravity rats were evaluated by immunohistochemical staining, quantitative RT-PCR, and Western blot analyses. The recruitment of monocytes in the common carotid artery of rats exposed to simulated microgravity was investigated by en face immunofluorescence staining and monocyte binding assays. Our results provided convincing evidence that there is an inflammatory response in the common carotid artery of rats exposed to simulated microgravity. Our work suggests that the inflammatory response may be a novel cellular mechanism that is responsible for the arterial remodeling that occurs during exposure to microgravity.
Assuntos
Doenças das Artérias Carótidas/metabolismo , Artéria Carótida Primitiva/metabolismo , Elevação dos Membros Posteriores/efeitos adversos , Animais , Peso Corporal , Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/patologia , Artéria Carótida Primitiva/patologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Selectina E/genética , Selectina E/metabolismo , Endotélio Vascular/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Monócitos/metabolismo , Ratos Sprague-Dawley , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Remodelação VascularRESUMO
INTRODUCTION: Irisin is closely related to type 2 diabetes mellitus (T2DM) and other metabolic diseases. It can improve the homeostasis of T2DM. MiR-133a-3p is decreased in the peripheral blood of patients with T2DM. Forkhead box protein O1 (FOXO1) is widely expressed in beta-cells and affects the occurrence of diabetes through transcriptional regulation and signalling pathway regulation. MATERIAL AND METHODS: The miR-133a-3p inhibitor was constructed to verify the effect of irisin on pyroptosis through miR-133a-3p. Next, we predicted the presence of targeted binding sequences between FOXO1 and miR-133a-3p by bioinformatics software, which was then confirmed with a double fluorescence assay. Finally, the FOXO1 overexpression vector was used to further verify the effect of irisin through the miR-133a-3p/FOXO1 axis. RESULTS: We first observed that irisin inhibited the protein levels of N-terminal gasdermin D (GSDMD-N) and cleaved caspase-1 and the secretion of interleukins (IL): IL-1beta and IL-18 in Min6 cells treated with high glucoes (HG). Irisin inhibited pyroptosis of Min6 cells treated with HG by reinforcing miR-133a-3p. Then, FOXO1 was validated to be the target gene of miR-133a. Both miR-133a-3p inhibitor and overexpression of FOXO1 restrained the force of irisin on pyroptosis in HG-induced Min6 cells. CONCLUSION: We explored the protective effect of irisin on HG-induced pyroptosis of islet b-cells in vitro and explained its mechanism of inhibiting pyroptosis through the miR-133a-3p/FOXO1 axis, to provide a theoretical basis for finding new molecular targets to delay beta-cell failure and the treatment of T2DM.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , MicroRNAs , Humanos , Piroptose , MicroRNAs/metabolismo , Fibronectinas , Células Secretoras de Insulina/metabolismo , Glucose , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismoRESUMO
Zein has enormous potential for application in biomedical field due to biodegradation and biocompatibility, we have recently prepared zein gel as a possible 3D printing ink. Our previous studies found that the pore structure in zein material can reduce early inflammation, promote the polarization of macrophages toward the M2 phenotype, and accelerate nerve regeneration. To further explore the role of zein in nerve repair, we used 4D printing technique to create nerve conduits with zein protein gel, and designed 2 types of tri-segment conduits with different degradation rates. Structural parts printed in support baths with higher water content show faster degradation rates than those printed in support baths with lower water content. The conduits that degraded quickly at both ends and slowly in the middle (CB75-CB40-CB75) and the conduits that degraded slowly at both ends and quickly in the middle (CB40-CB75-CB40) were 4D printed, respectively. Animal experiments suggest that the CB75-CB40-CB75 conduit is better for nerve repair, which may be because its degradation pattern can match to the pattern of nerve regeneration better. Our new strategy through 4D printing indicated that fine modulation in conduit degradation can affect efficacy of nerve repair significantly.
Assuntos
Tecido Nervoso , Zeína , Ratos , Animais , Ratos Sprague-Dawley , Zeína/química , Tinta , Nervo Isquiático/cirurgia , Nervo Isquiático/fisiologiaRESUMO
Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease associated with exposure to repetitive head impacts, which is susceptible in elderly people with declined mobility, athletes of full contact sports, military personnel and victims of domestic violence. It has been pathologically diagnosed in brain donors with a history of repetitive mild traumatic brain injury (rmTBI), but cannot be clinically diagnosed for a long time. By the continuous efforts by neuropathologists, neurologists and neuroscientists in recent 10 years, an expert consensus for the diagnostic framework of CTE was proposed in 2021 funded by the National Institute of Neurological Disorders and Stroke. The new consensus contributes to facilitating research in the field. However, it still needs to incorporate in vivo biomarkers to further refine and validate the clinical diagnostic criteria. From this, a single-center, observational cohort study has been being conducted by Tianjin Medical University General Hospital since 2021. As a pilot study of this clinical trial, the present research recruited 12 pairs of gender- and age-matched rmTBI patients with healthy subjects. Their blood samples were collected for exosome isolation, and multi-omics screening to explore potential diagnostic biomarkers in blood and its exosomes. The expression level of CHL1 protein, KIF2A mRNA, LIN7C mRNA, miR-297, and miR-1183 in serum and exosomes were found to be differentially expressed between groups. Besides, serum and exosomal CHL1, KIF2A, and miR-1183, as well as exosomal miR-297 were further verified as potential biomarkers for CTE by low-throughput assays. They are expected to contribute to establishing a novel set of CTE diagnostic signatures with classic neurodegenerative indicators in our future study, thereby updating the consensus diagnostic criteria for CTE by incorporating new evidence of the in vivo biomarkers.
RESUMO
1. The aim of the present study was to investigate the effects of simulated microgravity on the arterial dilatory responsiveness and L-arginine (L-Arg)-nitric oxide (NO)-cGMP pathway in the abdominal aorta of rats. 2. Twenty healthy male Sprague-Dawley were randomly divided into control and simulated microgravity groups. Rats in the simulated microgravity group were subjected to hindlimb unweighting (HU). After 4 weeks, arterial dilatory responsiveness was examined in vitro in isolated abdominal aortic rings. Western blotting was used to measure endothelial (e) and inducible (i) NO synthase (NOS) protein content. Total concentrations of nitrate and nitrite (NO(x)), the stable metabolites of NO, were determined by the chemiluminescence method. Nitric oxide synthase activity in the abdominal aorta was determined through the conversion of [(3)H]-L-Arg to [(3)H]-L-citrulline. 3. The data showed that the dilatory responses of the arterial rings to L-Arg and acetylcholine decreased in rats exposed to simulated microgravity, but the dilatory responses to sodium nitroprusside and 8-bromo-cGMP were similar in both simulated microgravity and control rats. The expression of eNOS and iNOS did not differ significantly between the two groups. The NO(x) concentration in the abdominal aorta of HU rats was significantly less than that in control rats. Nitric oxide synthase activity in the aorta decreased after 4 weeks of HU. 4. The data indicate that endothelium-dependent vasorelaxation in the abdominal aorta decreased due to 4 weeks of simulated microgravity in rats and that this impaired dilatory responsiveness may result from decreased NOS activity.
Assuntos
Aorta Abdominal/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase/metabolismo , Simulação de Ausência de Peso/estatística & dados numéricos , Acetilcolina/farmacologia , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/metabolismo , Aorta Abdominal/fisiologia , Arginina/farmacologia , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Masculino , Nitratos/metabolismo , Nitritos/metabolismo , Nitroprussiato/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Simulação de Ausência de Peso/métodosRESUMO
Parkinson's disease is a progressive neurodegenerative disorder characterized clinically by rigidity, akinesia, resting tremor and postural instability. It has recently been suggested that low frequency stimulation of the pedunculopontine nucleus (PPN) has a role in the therapy for Parkinsonism, particularly in gait disorder and postural instability. However, there is limited information about the mechanism of low frequency stimulation of the PPN on Parkinson's disease. The present study was to investigate the effect and mechanism of low frequency stimulation of the PPN on the firing rate of the ventrolateral thalamic nucleus (VL) in a rat model with unilateral 6-hydroxydopamine lesioning of the substantia nigra pars compacta. In vivo extracellular recording and microiontophoresis were adopted. The results showed that the firing rate of 60.71% VL neurons in normal rats and 59.57% VL neurons in 6-hydroxydopamine lesioned rats increased with low frequency stimulation of the PPN. Using microiontophoresis to VL neurons, we found the firing rate in VL neurons responded with either an increase or decrease in application of acetylcholine (ACh) in normal rats, whereas with a predominant decrease in M receptor antagonist atropine. Furthermore, the VL neurons were mainly inhibited by application of γ-aminobutyric acid (GABA) and excited by GABA(A) receptor antagonist bicuculline. Importantly, the VL neurons responding to ACh were also inhibited by application of GABA. We also found that the excitatory response of the VL neurons to the low frequency stimulation of the PPN was significantly reversed by microiontophoresis of atropine. These results demonstrate that cholinergic and GABAergic afferent nerve fibers may converge on the same VL neurons and they are involved in the effects of low frequency stimulation of the PPN, with ACh combining M(2) receptors on the presynaptic membrane of GABAergic afferents, which will inhibit the release of GABA in the VL and then improve the symptoms of Parkinson's disease.
Assuntos
Potenciais de Ação , Estimulação Elétrica , Doença de Parkinson Secundária/fisiopatologia , Núcleo Tegmental Pedunculopontino/fisiologia , Núcleos Ventrais do Tálamo/fisiologia , Acetilcolina/metabolismo , Animais , Fibras Colinérgicas/fisiologia , Masculino , Oxidopamina , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/terapia , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To establish an ex vivo model of myocardial ischemia reperfusion in tree shrews. METHODS: The Langendorff ex vivo heart perfusion system was used to establish the myocardial ischemia reperfusion model in tree shrews with different irrigation and reperfusion time settings. Alanine aminotransferase (ALT), aspartate transaminase (AST) and lactic dehydrogenase (LDH) levels were measured by enzyme-labeled immunosorbent assay, creatine kinase MB (CK-MB) was detected using immunosuppression method, and malondialdehyde was measured with thiobarbital staining method; the infarct size was measured using 2, 3, 5-triphenyltrazoliumchloride (TTC) method. RESULTS: Ischemia for 30 min and reperfusion for 30 and 60 min caused more significant increase in CK-MB and LDH levels in the perfusion fluid and also in the levels of ALT, CK-MB and AST in the myocardial tissue compared with other experimental settings (P<0.05), but these parameters were comparable between the former two settings (P>0.05). The mean heart rate in 30-min ischemia with 60-min reperfusion group was obviously lower than that in continuous reperfusion group, 15-min ischemia with 30-min reperfusion group and 30-min ischemia with 30-min reperfusion group (P<0.05), and the heart rate was similar between the latter 3 groups (P>0.05). ECG analysis showed that the mean heart rate in 30-min ischemia with 30-min reperfusion group was closer to the physiological heart rate of tree shrews. CONCLUSION: We successfully established an ex vivo myocardial ischemia reperfusion model using tree shrews, and ischemia for 30 min followed by reperfusion for 30 min is the optimal experimental setting.
Assuntos
Modelos Animais de Doenças , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Alanina Transaminase/análise , Animais , Aspartato Aminotransferases/análise , Creatina Quinase Forma MB/análise , L-Lactato Desidrogenase/análise , Malondialdeído/análise , Miocárdio , TupaiidaeRESUMO
BACKGROUND: Athletes, military personnel and mobility-declined elderly people are prone to repetitive mild traumatic brain injury (rmTBI). The injury does not cause acute pathological changes, but leads to chronic neurodegeneration, long-term cognitive dysfunction and even chronic traumatic encephalopathy (CTE). Many existing rmTBI animal models reported uncontrollable adverse effects and long experiment period. Therefore, an improved model needs to be designed. NEW METHOD: Our rmTBI mouse model is a modification of the closed head injury method using electronic controlled cortical impact system. Discontinuous 4 impacts with 48-h interval were performed. A key facet of the model is the use of our designed molded acrylic cast and concave metal disc (as a helmet). They could scatter and transmit hitting power to the whole brain, thus produced a mild diffused injury. The procedure does not require scalp incision or craniotomy, which allows the impacting to be completed in 2 min. RESULTS: Our model did not induce acute macroscopic brain damage and brain edema. It could lead to sustained neuroinflammation and chronic neurodegeneration in injured brain, and resulted in cognitive dysfunction within 5 weeks post-injury. COMPARISON WITH EXISTING METHODS: Previously reported adverse effects including skull fractures, hemorrhage and brain tissue loss were not observed in our model. An experiment period of 5 weeks was allowed for observing chronic neurodegeneration and cognitive dysfunction. CONCLUSIONS: Our model is beneficial to use for simplicity, reproducibility and time saver. It could serve as a platform for research on the pathogenesis, diagnosis and potential therapeutics for rmTBI and CTE.
Assuntos
Concussão Encefálica/fisiopatologia , Encefalopatia Traumática Crônica/fisiopatologia , Modelos Animais de Doenças , Animais , Comportamento Animal , Encéfalo/patologia , Concussão Encefálica/patologia , Concussão Encefálica/psicologia , Encefalopatia Traumática Crônica/patologia , Encefalopatia Traumática Crônica/psicologia , Masculino , Camundongos Endogâmicos C57BL , Neurônios/patologia , Reprodutibilidade dos Testes , Teste de Desempenho do Rota-RodRESUMO
We report a strategy for programmed DNA self-assembly that is favorable in terms of both thermodynamics and kinetics. In a previous study, it has been demonstrated that DNA self-assembly is primarily driven by thermodynamics and the assembly kinetics is not considered. To reach such stable states at equilibria, prolonged annealing duration is needed. In addition, there are cases where the desired structures could not compete with alternative structures. For example, a single-stranded DNA with a palindromic sequence quickly folds into a one-stranded hairpin instead of forming a two-stranded DNA duplex. Given that most of the DNA tiles are multi-stranded complexes, the kinetic trap represents a challenge to DNA self-assembly. To overcome this problem, we have developed a one-stranded motif that can intramolecularly and quickly fold from a single DNA strand and can be programmed to assemble into a range of nanostructures, including a one-dimensional (1D) ladder, a 1D chain, a two-dimensional (2D) array, and a three-dimensional (3D) triangular prism. All structures have been characterized by polyacrylamide gel electrophoresis (PAGE) and atomic force microscopy (AFM) imaging.
Assuntos
DNA/química , Nanoestruturas/química , Conformação de Ácido Nucleico , DNA de Cadeia Simples , Eletroforese em Gel de Poliacrilamida , Microscopia de Força AtômicaRESUMO
Rational, de novo design of RNA nanostructures can potentially integrate a wide array of structural and functional diversities. Such nanostructures have great promises in biomedical applications. Despite impressive progress in this field, all RNA building blocks (or tiles) reported so far are not geometrically well defined. They are generally flexible and can only assemble into a mixture of complexes with different sizes. To achieve defined structures, multiple tiles with different sequences are needed. In this study, we design an RNA tile that can homo-oligomerize into a uniform RNA nanostructure. The designed RNA nanostructure is characterized by gel electrophoresis, atomic force microscopy and cryogenic electron microscopy imaging. We believe that development along this line would help RNA nanotechnology to reach the structural control that is currently associated with DNA nanotechnology.
Assuntos
Nanoestruturas/química , Nanotecnologia/métodos , RNA/química , DNA/químicaRESUMO
Previous studies have demonstrated inconsistent roles of Rho kinase (ROCK) in the decreased vasoconstriction of rat hindquarter vessels induced by hindlimb unweighting (HU). The present study was designed to determine the unclear role of ROCK in the mediation of HU-induced decreased femoral arterial vasoconstriction. 28-day HU rat was adopted as the animal model. With or without Y-27632, a ROCK inhibitor, isometric force of femoral artery was measured. The expression of ROCK and its effects on downstream targets were also examined. Results showed that (1) HU caused a significant decrease of the phenylephrine (PE)-evoked and potassium chloride (KCl)-evoked femoral arterial vasoconstriction (P < 0.05), confirming the functional findings by previous studies. (2) Inhibition of ROCK with Y-27632 produced an equal reduction of the vasoconstriction in CON and HU. (3) HU significantly decreased ROCK II expression and the effects of ROCK on myosin light-chain phosphatase (MLCP) and MLC (P < 0.05), but increased p65 nuclear translocation (P < 0.05) and inducible nitric oxide synthase (iNOS) expression (P < 0.05). (4) HU significantly (P < 0.05) increased NO production in femoral arteries, with Y-27632 significantly (P < 0.01) amplifying this effect. These findings have revealed that 28-day HU reduced the expression and effects of ROCK on downstream targets both directly (MLCP and MLC) and possibly indirectly (NF-κB/iNOS/NO pathway) related to vasoconstriction in femoral arteries.