RESUMEN
The assessment of three-dimensional (3D) brain cytoarchitecture at a cellular resolution remains a great challenge in the field of neuroscience and constant development of imaging techniques has become crucial, particularly when it comes to offering direct and clear obtention of data from macro to nano scales. Magnetic resonance imaging (MRI) and electron or optical microscopy, although valuable, still face some issues such as the lack of contrast and extensive sample preparation protocols. In this context, x-ray microtomography (µCT) has become a promising non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens. It is a new supplemental method to be explored for deciphering the cytoarchitecture and connectivity of the brain. This review aims to bring together published works using x-ray µCT in neurobiology in order to discuss the achievements made so far and the future of this technique for neuroscience.
RESUMEN
There is a lack of information correlating low adiposity with hypertension experienced by Spontaneous Hypertensive Rats (SHR) or overweight and normotension in Wistar-Kyoto (WKY). We aimed to investigate this lipodystrophy phenomenon by measuring fluorescence lifetime (FLIM), optical redox ratio (ORR), serum levels of hypothalamic-pituitary-adrenal (HPA) and/or hypothalamic-pituitary-thyroid (HPT) hormones axes between Wistar, WKY and SHR before and after establishment of hypertension. Under high blood pressure, we evaluated serum adipokines. Brown adipose tissue was characterized as lower ORR and shorter FLIM compared to white adipose tissue. HPT axis showed a crucial role in the SHR adipose tissue configuration by attenuating whitening. The increased adiposity in WKY may act as a preventive agent for hypertension, since SHR, with low adiposity, establishes the disease. The hypertensive environment can highlight key adipokines that may result in new therapeutic approaches to the treatment of adiposity dysfunctions and hypertension.
Asunto(s)
Tejido Adiposo Pardo/fisiología , Tejido Adiposo/fisiología , Hipertensión , Lipodistrofia , Adipoquinas/sangre , Tejido Adiposo/diagnóstico por imagen , Tejido Adiposo/metabolismo , Tejido Adiposo Pardo/diagnóstico por imagen , Animales , Presión Sanguínea/fisiología , Hipertensión/complicaciones , Hipertensión/diagnóstico por imagen , Hipertensión/metabolismo , Hipertensión/fisiopatología , Sistema Hipotálamo-Hipofisario/diagnóstico por imagen , Sistema Hipotálamo-Hipofisario/fisiología , Lipodistrofia/diagnóstico por imagen , Lipodistrofia/etiología , Lipodistrofia/fisiopatología , Masculino , Microscopía Fluorescente/métodos , Oxidación-Reducción , Sistema Hipófiso-Suprarrenal/diagnóstico por imagen , Sistema Hipófiso-Suprarrenal/fisiología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Glándula Tiroides/diagnóstico por imagen , Glándula Tiroides/fisiologíaRESUMEN
The spread of microtomography as a tool for visualization of soft tissues has had a significant impact on a better understanding of complex biological systems. This technique allows a detailed three-dimensional quantitative view of the specimen to be obtained, correlating its morphological organization with its function, providing valuable insights on the functionality of the tissue. Regularly overlooked, but of great importance, proper sample mounting and preparation are fundamental for achieving the highest possible image quality even for the high-resolution imaging systems currently under development. Here, a quantitative analysis compares some of the most common sample-mounting strategies used for synchrotron-based X-ray microtomography of soft tissues: alcoholic-immersion, paraffin-embedding and critical-point drying. These three distinct sample-mounting strategies were performed on the same specimen in order to investigate their impact on sample morphology regardless of individual sample variation. In that sense, the alcoholic-immersion strategy, although causing less shrinkage to the tissue, proved to be the most unsuitable approach for a high-throughput high-resolution imaging experiment due to sample drifting. Also, critical-point drying may present some interesting advantages regarding image quality but is also incompatible with a high-throughput experiment. Lastly, paraffin-embedding is shown to be the most suitable strategy for current soft tissue microtomography experiments. Such detailed analysis of biological sample-mounting strategies for synchrotron-based X-ray microtomography are expected to offer valuable insights on the best approach for using this technique for 3D imaging of soft tissues and following morphometric analysis.
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Alcoholic liver disease (ALD) is a highly prevalent spectrum of pathologies caused by alcohol overconsumption. Morbidity and mortality related to ALD are increasing worldwide, thereby demanding strategies for early diagnosis and detection of ALD predisposition. A potential candidate as a marker for ALD susceptibility is the transcription factor nuclear factor erythroid-related factor 2 (Nrf2), codified by the nuclear factor erythroid 2-related factor 2 gene (NFE2L2). Nrf2 regulates expression of proteins that protect against oxidative stress and inflammation caused by alcohol overconsumption. Here, we assessed genetic variants of NFE2L2 for association with ALD. Specimens from patients diagnosed with cirrhosis caused by ALD were genotyped for three NFE2L2 single nucleotide polymorphisms (SNP) (SNPs: rs35652124, rs4893819, and rs6721961). Hematoxylin & eosin and immunohistochemistry were performed to determine the inflammatory score and Nrf2 expression, respectively. SNPs rs4893819 and rs6721961 were not specifically associated with ALD, but analysis of SNP rs35652124 suggested that this polymorphism predisposes to ALD. Furthermore, SNP rs35652124 was associated with a lower level of Nrf2 expression. Moreover, liver samples from ALD patients with this polymorphism displayed more severe inflammatory activity. Together, these findings provide evidence that the SNP rs35652124 variation in the Nrf2-encoding gene NFE2L2 is a potential genetic marker for susceptibility to ALD.
Asunto(s)
Predisposición Genética a la Enfermedad , Cirrosis Hepática Alcohólica/genética , Factor 2 Relacionado con NF-E2/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Adulto , Estudios de Casos y Controles , Etanol/farmacología , Femenino , Expresión Génica , Hepacivirus/crecimiento & desarrollo , Hepacivirus/patogenicidad , Hepatitis C/patología , Hepatitis C/virología , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Alcohólica/metabolismo , Cirrosis Hepática Alcohólica/patología , Cirrosis Hepática Alcohólica/cirugía , Trasplante de Hígado , Masculino , Persona de Mediana Edad , Estrés OxidativoRESUMEN
In chronic schistosomiasis, liver fibrosis is linked to portal hypertension, which is a condition associated with high mortality and morbidity. High mobility group box 1 (HMGB1) was originally described as a nuclear protein that functions as a structural co-factor in transcriptional regulation. However, HMGB1 can also be secreted into the extracellular milieu under appropriate signal stimulation. Extracellular HMGB1 acts as a multifunctional cytokine that contributes to infection, injury, inflammation, and immune responses by binding to specific cell-surface receptors. HMGB1 is involved in fibrotic diseases. From a clinical perspective, HMGB1 inhibition may represent a promising therapeutic approach for treating tissue fibrosis. In this study, we demonstrate elevated levels of HMGB1 in the sera in experimental mice or in patients with schistosomiasis. Using immunohistochemistry, we demonstrated that HMGB1 trafficking in the hepatocytes of mice suffering from acute schistosomiasis was inhibited by Glycyrrhizin, a well-known HMGB1 direct inhibitor, as well as by DIC, a novel and potential anti-HMGB1 compound. HMGB1 inhibition led to significant downregulation of IL-6, IL4, IL-5, IL-13, IL-17A, which are involved in the exacerbation of the immune response and liver fibrogenesis. Importantly, infected mice that were treated with DIC or GZR to inhibit HMGB1 pro-inflammatory activity showed a significant increase in survival and a reduction of over 50% in the area of liver fibrosis. Taken together, our findings indicate that HMGB1 is a key mediator of schistosomotic granuloma formation and liver fibrosis and may represent an outstanding target for the treatment of schistosomiasis.
Asunto(s)
Granuloma , Proteína HMGB1/inmunología , Cirrosis Hepática , Hígado , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni , Animales , Citocinas/inmunología , Femenino , Granuloma/inmunología , Granuloma/parasitología , Granuloma/patología , Humanos , Hígado/inmunología , Hígado/parasitología , Hígado/patología , Cirrosis Hepática/inmunología , Cirrosis Hepática/parasitología , Cirrosis Hepática/patología , Masculino , Ratones Endogámicos BALB C , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/patologíaRESUMEN
The assessment of neuronal number, spatial organization and connectivity is fundamental for a complete understanding of brain function. However, the evaluation of the three-dimensional (3D) brain cytoarchitecture at cellular resolution persists as a great challenge in the field of neuroscience. In this context, X-ray microtomography has shown to be a valuable non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens, arisen as a new method for deciphering the cytoarchitecture and connectivity of the brain. In this work we present a method for imaging whole neurons in the brain, combining synchrotron-based X-ray microtomography with the Golgi-Cox mercury-based impregnation protocol. In contrast to optical 3D techniques, the approach shown here does neither require tissue slicing or clearing, and allows the investigation of several cells within a 3D region of the brain.
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Encéfalo/citología , Imagenología Tridimensional/métodos , Neuronas , Microtomografía por Rayos X/métodos , Animales , Encéfalo/diagnóstico por imagen , Imagenología Tridimensional/instrumentación , Cloruro de Mercurio/química , Ratones , Tinción con Nitrato de Plata/métodos , Sincrotrones , Fijación del Tejido/métodos , Microtomografía por Rayos X/instrumentaciónRESUMEN
Hepatocyte proliferation during liver regeneration is a well-coordinated process regulated by the activation of several growth factor receptors, including the insulin receptor (IR). The IR can be localized in part to cholesterol-enriched membrane microdomains, but the role of such domains in insulin-mediated events in hepatocytes is not known. We investigated whether partitioning of IRs into cholesterol-enriched membrane rafts is important for the mitogenic effects of insulin in the hepatic cells. IR and lipid rafts were labeled in HepG2 cells and primary rat hepatocytes. Membrane cholesterol was depleted in vitro with metyl-ß-cyclodextrin (MßCD) and in vivo with lovastatin. Insulin-induced calcium (Ca2+) signals studies were examined in HepG2 cells and in freshly isolated rat hepatocytes as well as in whole liver in vivo by intravital confocal imaging. Liver regeneration was studied by 70% partial hepatectomy (PH), and hepatocyte proliferation was assessed by PCNA staining. A subpopulation of IR was found in membrane microdomains enriched in cholesterol. Depletion of cholesterol from plasma membrane resulted in redistribution of the IR along the cells, which was associated with impaired insulin-induced nuclear Ca2+ signals, a signaling event that regulates hepatocyte proliferation. Cholesterol depletion also led to ERK1/2 hyper-phosphorylation. Lovastatin administration to rats decreased hepatic cholesterol content, disrupted lipid rafts and decreased insulin-induced Ca2+ signaling in hepatocytes, and delayed liver regeneration after PH. Therefore, membrane cholesterol content and lipid rafts integrity showed to be important for the proliferative effects of insulin in hepatic cells. NEW & NOTEWORTHY One of insulin's actions is to stimulate liver regeneration. Here we show that a subpopulation of insulin receptors is in a specialized cholesterol-enriched region of the cell membrane and this subfraction is important for insulin's proliferative effects.
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Calcio/metabolismo , Colesterol/metabolismo , Hepatocitos/metabolismo , Insulina/metabolismo , Regeneración Hepática/fisiología , Microdominios de Membrana/fisiología , Receptor de Insulina/metabolismo , Animales , Proliferación Celular/fisiología , Ratas , Transducción de Señal/fisiologíaRESUMEN
Increases in nuclear calcium concentration generate specific biological outcomes that differ from those resulting from increased cytoplasmic calcium. Nuclear calcium effects on tumor cell proliferation are widely appreciated; nevertheless, its involvement in other steps of tumor progression is not well understood. Therefore, we evaluated whether nuclear calcium is essential in other additional stages of tumor progression, including key steps associated with the formation of the primary tumor or with the metastatic cascade. We found that nuclear calcium buffering impaired 4T1 triple negative breast cancer growth not just by decreasing tumor cell proliferation, but also by enhancing tumor necrosis. Moreover, nuclear calcium regulates tumor angiogenesis through a mechanism that involves the upregulation of the anti-angiogenic C-X-C motif chemokine 10 (CXCL10-IP10). In addition, nuclear calcium buffering regulates breast tumor cell motility, culminating in less cell invasion, likely due to enhanced vinculin expression, a focal adhesion structural protein. Together, our results show that nuclear calcium is essential for triple breast cancer angiogenesis and cell migration and can be considered as a promising strategic target for triple negative breast cancer therapy.
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Señalización del Calcio , Inositol 1,4,5-Trifosfato/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Núcleo Celular/metabolismo , Proliferación Celular , Quimiocina CXCL10/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos BALB C , Neovascularización Patológica/genética , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND: The angiotensin-I converting enzyme (ACE) plays a central role in the renin-angiotensin system, acting by converting the hormone angiotensin-I to the active peptide angiotensin-II (Ang-II). More recently, ACE was shown to act as a receptor for Ang-II, and its expression level was demonstrated to be higher in melanoma cells compared to their normal counterparts. However, the function that ACE plays as an Ang-II receptor in melanoma cells has not been defined yet. AIM: Therefore, our aim was to examine the role of ACE in tumor cell proliferation and migration. RESULTS: We found that upon binding to ACE, Ang-II internalizes with a faster onset compared to the binding of Ang-II to its classical AT1 receptor. We also found that the complex Ang-II/ACE translocates to the nucleus, through a clathrin-mediated process, triggering a transient nuclear Ca2+ signal. In silico studies revealed a possible interaction site between ACE and phospholipase C (PLC), and experimental results in CHO cells, demonstrated that the ß3 isoform of PLC is the one involved in the Ca2+ signals induced by Ang-II/ACE interaction. Further studies in melanoma cells (TM-5) showed that Ang-II induced cell proliferation through ACE activation, an event that could be inhibited either by ACE inhibitor (Lisinopril) or by the silencing of ACE. In addition, we found that stimulation of ACE by Ang-II caused the melanoma cells to migrate, at least in part due to decreased vinculin expression, a focal adhesion structural protein. CONCLUSION: ACE activation regulates melanoma cell proliferation and migration.
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Angiotensina II/metabolismo , Núcleo Celular/metabolismo , Melanoma/enzimología , Peptidil-Dipeptidasa A/metabolismo , Fosfolipasa C beta/metabolismo , Vinculina/metabolismo , Animales , Células CHO , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Cricetulus , Humanos , Lisinopril/farmacología , Melanoma/genética , Melanoma/metabolismo , Peptidil-Dipeptidasa A/genética , Transporte de ProteínasRESUMEN
AIM: To investigate the presynaptic effects of propofol, a short-acting intravenous anesthetic, in the frog neuromuscular junction. METHODS: Frog cutaneous pectoris nerve muscle preparations were prepared. A fluorescent tool (FM1-43) was used to visualize the effect of propofol on synaptic vesicle exocytosos in the frog neuromuscular junction. RESULTS: Low concentrations of propofol, ranging from 10 to 25 µmol/L, enhanced spontaneous vesicle exocytosis monitored by FM1-43 in a Ca(2+)-dependent and Na(+)-independent fashion. Higher concentrations of propofol (50, 100, and 200 µmol/L) had no effect on spontaneous exocytosis. By contrast, higher concentrations of propofol inhibited the Na(+)-dependent exocytosis evoked by 4-aminopyridine but did not affect the Na(+)-independent exocytosis evoked by KCl. This action was similar and non-additive with that observed by tetrodotoxin, a Na(+) channel blocker. CONCLUSION: Our data suggest that propofol has a dose-dependent presynaptic effect at the neuromuscular transmission which may help to understand some of the clinical effects of this agent on neuromuscular function.