RESUMEN
Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.
Asunto(s)
Calcio/fisiología , Técnicas Electrofisiológicas Cardíacas , Función Ventricular , Ventrículos Cardíacos , Humanos , Técnicas In Vitro , Potenciales de la Membrana , MicrotomíaRESUMEN
Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.
Asunto(s)
Enfermedades Pulmonares/patología , Pulmón/patología , Microtomía/métodos , Manejo de Especímenes/métodos , Animales , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Humanos , Fibrosis Pulmonar Idiopática/patología , Neoplasias Pulmonares/patología , Ratones , Enfermedad Pulmonar Obstructiva Crónica/patologíaRESUMEN
In addition to their support role in neurotransmitter and ion buffering, astrocytes directly regulate neurotransmission at synapses via local bidirectional signaling with neurons. Here, we reveal a form of neuronal-astrocytic signaling that transmits retrograde dendritic signals to distal upstream neurons in order to activate recurrent synaptic circuits. Norepinephrine activates α1 adrenoreceptors in hypothalamic corticotropin-releasing hormone (CRH) neurons to stimulate dendritic release, which triggers an astrocytic calcium response and release of ATP; ATP stimulates action potentials in upstream glutamate and GABA neurons to activate recurrent excitatory and inhibitory synaptic circuits to the CRH neurons. Thus, norepinephrine activates a retrograde signaling mechanism in CRH neurons that engages astrocytes in order to extend dendritic volume transmission to reach distal presynaptic glutamate and GABA neurons, thereby amplifying volume transmission mediated by dendritic release.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Astrocitos/efectos de los fármacos , Dendritas/efectos de los fármacos , Neuronas GABAérgicas/efectos de los fármacos , Norepinefrina/farmacología , Transmisión Sináptica/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Astrocitos/metabolismo , Astrocitos/ultraestructura , Comunicación Celular , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Hormona Liberadora de Corticotropina/genética , Hormona Liberadora de Corticotropina/metabolismo , Dendritas/metabolismo , Dendritas/ultraestructura , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/ultraestructura , Regulación de la Expresión Génica , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Hipotálamo/ultraestructura , Masculino , Ratones , Ratones Transgénicos , Microtomía , Receptores de Corticotropina/genética , Receptores de Corticotropina/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Técnicas de Cultivo de Tejidos , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.
Asunto(s)
Proteína Similar al Receptor de Calcitonina/genética , Endocitosis/efectos de los fármacos , Endosomas/metabolismo , Nocicepción/fisiología , Dolor/fisiopatología , Transmisión Sináptica/efectos de los fármacos , Antagonistas Adrenérgicos/farmacología , Animales , Péptido Relacionado con Gen de Calcitonina/farmacología , Proteína Similar al Receptor de Calcitonina/antagonistas & inhibidores , Proteína Similar al Receptor de Calcitonina/metabolismo , Capsaicina/antagonistas & inhibidores , Capsaicina/farmacología , Colestanoles/farmacología , Clatrina/antagonistas & inhibidores , Clatrina/genética , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Endosomas/efectos de los fármacos , Formaldehído/antagonistas & inhibidores , Formaldehído/farmacología , Adyuvante de Freund/antagonistas & inhibidores , Adyuvante de Freund/farmacología , Regulación de la Expresión Génica , Inyecciones Espinales , Masculino , Ratones , Microtomía , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nocicepción/efectos de los fármacos , Dolor/inducido químicamente , Dolor/genética , Dolor/prevención & control , Fragmentos de Péptidos/farmacología , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Ratas , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Phosphatidylcholine is the main phospholipid present in cell membranes and in lipoproteins, and can interfere with various biological processes. This lipid also has antioxidant activity, and protects against damage caused by free radicals under conditions of ischemia/reperfusion. Therefore, the present study was designed to evaluate toxicogenetic damage caused by twisting of the spermatic cord in ischemia/reperfusion, and whether phosphatidylcholine plays a role in conditions of ischemia/reperfusion in preclinical trials. The results indicate that spermatic cord torsion does not cause genotoxic damage or mutagenesis. A dose of 300 mg/kg of phosphatidylcholine is toxic and is thus not recommended. However, a dose of 150 mg/kg does not promote toxicogenetic damage, and though it does not statistically prevent tissue damage occurring from lack of oxygenation and nutrition of testicular cells, it has a tendency to reduce this damage. Therefore, this research suggests that further studies should be conducted to clarify this tendency and to provide a better explanation of the possible therapeutic effects of phosphatidylcholine in cytoprotection of germ cells affected by ischemia/reperfusion.
Asunto(s)
Antioxidantes/farmacología , Fosfatidilcolinas/farmacología , Daño por Reperfusión/tratamiento farmacológico , Cordón Espermático/efectos de los fármacos , Testículo/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Ensayo Cometa , Evaluación Preclínica de Medicamentos , Histocitoquímica , Inyecciones Intraperitoneales , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Pruebas de Micronúcleos , Microtomía , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Cordón Espermático/irrigación sanguínea , Cordón Espermático/metabolismo , Cordón Espermático/patología , Testículo/irrigación sanguínea , Testículo/metabolismo , Testículo/patología , Torsión MecánicaRESUMEN
Cryofixation and freeze-substitution techniques provide excellent preservation of plant ultrastructure. The advantage of cryofixation is not only in structural preservation, as seen in the smooth plasma membrane, but also in the speed in arresting cell activity. Immunoelectron microscopy reveals the subcellular localization of molecules within cells. Immunolabeling in combination with cryofixation and freeze-substitution techniques provides more detailed information on the immunoelectron-microscopic localization of molecules in the plant cell than can be obtained from chemically fixed tissues. Here, we introduce methods for immunoelectron microscopy of cryofixed and freeze-substituted plant tissues.
Asunto(s)
Criopreservación/métodos , Substitución por Congelación/métodos , Inmunohistoquímica/métodos , Cebollas/ultraestructura , Células Vegetales/ultraestructura , Adhesión del Tejido/métodos , Anticuerpos/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Resinas Epoxi/química , Fijadores/química , Expresión Génica , Glutaral/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Microscopía Inmunoelectrónica/métodos , Microtomía , Cebollas/metabolismo , Células Vegetales/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tallos de la Planta/metabolismo , Tallos de la Planta/ultraestructura , Populus/metabolismo , Populus/ultraestructura , Semillas/metabolismo , Semillas/ultraestructura , Coloración y Etiquetado/métodos , Fijación del Tejido/métodos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Peroxisomal proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor whose agonist, rosiglitazone has a neuroprotective effect to hippocampal neurons in pilocarpine-induced seizures. Hippocampal slice preparations treated in Mg2+ free medium can induce ictal and interictal-like epileptiform discharges, which is regarded as an in vitro model of N-methyl-D-aspartate (NMDA) receptor-mediated temporal lobe epilepsy (TLE). We applied rosiglitazone in hippocampal slices treated in Mg2+ free medium. The effects of rosiglitazone on hippocampal CA1-Schaffer collateral synaptic transmission were tested. We also examined the neuroprotective effect of rosiglitazone toward NMDA excitotoxicity on cultured hippocampal slices. Application of 10 µM rosiglitazone significantly suppressed amplitude and frequency of epileptiform discharges in CA1 neurons. Pretreatment with the PPARγ antagonist GW9662 did not block the effect of rosiglitazone on suppressing discharge frequency, but reverse the effect on suppressing discharge amplitude. Application of rosiglitazone suppressed synaptic transmission in the CA1-Schaffer collateral pathway. By miniature excitatory-potential synaptic current (mEPSC) analysis, rosiglitazone significantly suppressed presynaptic neurotransmitter release. This phenomenon can be reversed by pretreating PPARγ antagonist GW9662. Also, rosiglitazone protected cultured hippocampal slices from NMDA-induced excitotoxicity. The protective effect of 10 µM rosiglitazone was partially antagonized by concomitant high dose GW9662 treatment, indicating that this effect is partially mediated by PPARγ receptors. In conclusion, rosiglitazone suppressed NMDA receptor-mediated epileptiform discharges by inhibition of presynaptic neurotransmitter release. Rosiglitazone protected hippocampal slice from NMDA excitotoxicity partially by PPARγ activation. We suggest that rosiglitazone could be a potential agent to treat patients with TLE.
Asunto(s)
Región CA1 Hipocampal/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Ácido Glutámico/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Tiazolidinedionas/farmacología , Potenciales de Acción/efectos de los fármacos , Anilidas/farmacología , Animales , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Medios de Cultivo/química , Medios de Cultivo/farmacología , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/metabolismo , Epilepsia del Lóbulo Temporal/patología , Regulación de la Expresión Génica , Magnesio/farmacología , Microtomía , Modelos Biológicos , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/antagonistas & inhibidores , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Rosiglitazona , Convulsiones/tratamiento farmacológico , Convulsiones/genética , Convulsiones/metabolismo , Convulsiones/patología , Transmisión Sináptica/efectos de los fármacos , Tiazolidinedionas/antagonistas & inhibidores , Técnicas de Cultivo de TejidosRESUMEN
Motor skill learning is characterized by improved performance and reduced motor variability. The neural mechanisms that couple skill level and variability, however, are not known. The zebra finch, a songbird, presents a unique opportunity to address this question because production of learned song and induction of vocal variability are instantiated in distinct circuits that converge on a motor cortex analogue controlling vocal output. To probe the interplay between learning and variability, we made intracellular recordings from neurons in this area, characterizing how their inputs from the functionally distinct pathways change throughout song development. We found that inputs that drive stereotyped song-patterns are strengthened and pruned, while inputs that induce variability remain unchanged. A simple network model showed that strengthening and pruning of action-specific connections reduces the sensitivity of motor control circuits to variable input and neural 'noise'. This identifies a simple and general mechanism for learning-related regulation of motor variability.
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Pinzones/fisiología , Aprendizaje/fisiología , Red Nerviosa/fisiología , Neuronas/fisiología , Vocalización Animal/fisiología , Estimulación Acústica , Animales , Ganglios Basales/citología , Ganglios Basales/fisiología , Masculino , Potenciales de la Membrana/fisiología , Microtomía , Corteza Motora/citología , Corteza Motora/fisiología , Redes Neurales de la Computación , Neuronas/citología , Técnicas de Placa-Clamp , Técnicas de Cultivo de TejidosRESUMEN
The motor cortex (M1) is classically considered an agranular area, lacking a distinct layer 4 (L4). Here, we tested the idea that M1, despite lacking a cytoarchitecturally visible L4, nevertheless possesses its equivalent in the form of excitatory neurons with input-output circuits like those of the L4 neurons in sensory areas. Consistent with this idea, we found that neurons located in a thin laminar zone at the L3/5A border in the forelimb area of mouse M1 have multiple L4-like synaptic connections: excitatory input from thalamus, largely unidirectional excitatory outputs to L2/3 pyramidal neurons, and relatively weak long-range corticocortical inputs and outputs. M1-L4 neurons were electrophysiologically diverse but morphologically uniform, with pyramidal-type dendritic arbors and locally ramifying axons, including branches extending into L2/3. Our findings therefore identify pyramidal neurons in M1 with the expected prototypical circuit properties of excitatory L4 neurons, and question the traditional assumption that motor cortex lacks this layer.
Asunto(s)
Potenciales de Acción/fisiología , Corteza Motora/fisiología , Células Piramidales/fisiología , Sinapsis/fisiología , Potenciales Sinápticos/fisiología , Adenoviridae/genética , Animales , Axones/fisiología , Axones/ultraestructura , Dendritas/fisiología , Dendritas/ultraestructura , Colorantes Fluorescentes , Vectores Genéticos , Ratones , Microesferas , Microtomía , Corteza Motora/ultraestructura , Células Piramidales/ultraestructura , Técnicas Estereotáxicas , Sinapsis/ultraestructura , Transmisión Sináptica , Tálamo/fisiología , Tálamo/ultraestructura , Técnicas de Cultivo de TejidosRESUMEN
Extracellular superoxide dismutase (EC-SOD) is an isoform of SOD normally found both intra- and extra-cellularly and accounting for most SOD activity in blood vessels. Here we explored the role of EC-SOD in protecting against brain damage induced by chronic hypoxia. EC-SOD Transgenic mice, were exposed to hypoxia (FiO2.1%) for 10 days (H-KI) and compared to transgenic animals housed in room air (RA-KI), wild type animals exposed to hypoxia (H-WT or wild type mice housed in room air (RA-WT). Overall brain metabolism evaluated by positron emission tomography (PET) showed that H-WT mice had significantly higher uptake of 18FDG in the brain particularly the hippocampus, hypothalamus, and cerebellum. H-KI mice had comparable uptake to the RA-KI and RA-WT groups. To investigate the functional state of the hippocampus, electrophysiological techniques in ex vivo hippocampal slices were performed and showed that H-KI had normal synaptic plasticity, whereas H-WT were severely affected. Markers of oxidative stress, GFAP, IBA1, MIF, and pAMPK showed similar values in the H-KI and RA-WT groups, but were significantly increased in the H-WT group. Caspase-3 assay and histopathological studies showed significant apoptosis/cell damage in the H-WT group, but no significant difference in the H-KI group compared to the RA groups. The data suggest that EC-SOD has potential prophylactic and therapeutic roles in diseases with compromised brain oxygenation.
Asunto(s)
Isquemia Encefálica/genética , Expresión Génica , Hipoxia/genética , Superóxido Dismutasa/genética , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Apoptosis , Biomarcadores , Isquemia Encefálica/enzimología , Isquemia Encefálica/patología , Isquemia Encefálica/prevención & control , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Cerebelo/metabolismo , Cerebelo/patología , Fluorodesoxiglucosa F18/metabolismo , Proteína Ácida Fibrilar de la Glía , Hipocampo/metabolismo , Hipocampo/patología , Hipotálamo/metabolismo , Hipotálamo/patología , Hipoxia/enzimología , Hipoxia/patología , Hipoxia/prevención & control , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microtomía , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Tomografía de Emisión de Positrones , Superóxido Dismutasa/metabolismo , Técnicas de Cultivo de Tejidos , TransgenesRESUMEN
We found that the mucilaginous substance of the Assyrian plum, Cordia myxa, can be used as an adhesive for attaching sections of animal tissues to slides. Unlike Mayer's albumen, this material left no stainable residue and had no noticeable effect on the histological structure of the tissue sections. The mucilaginous substance of C. myxa is a useful and inexpensive alternative to standard adhesives.
Asunto(s)
Cordia/química , Microtomía/métodos , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Semillas/química , Adhesivos Tisulares/química , Irak , Microtomía/economía , Extractos Vegetales/economía , Adhesivos Tisulares/economíaRESUMEN
Pollen grain morphology has been widely used in the classification of the Acanthaceae, where external pollen wall features have proved useful in determining relationships between taxa. Although detailed information has been accumulated using light microscopy, transmission electron microscopy and scanning electron microscopy (SEM) techniques, internal pollen wall features lack investigation and the techniques are cumbersome. A new technique involving precise cross sectioning or slicing of pollen grains at a selected position for examining wall ultrastructure, using a focused ion beam-scanning electron microscope (FIB-SEM), has been explored and promising results have been obtained. The FIB-SEM offers a good technique for reliable, high resolution, three-dimensional (3D) viewing of the internal structure of the pollen grain wall.
Asunto(s)
Acanthaceae/ultraestructura , Pared Celular/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Microtomía/métodos , Polen/ultraestructura , Imagenología Tridimensional/métodosRESUMEN
BACKGROUND: Smear layer is a negative factor which prevents adhesion of the filling material to the dentinal walls. Recent advances in dental research have incorporated lasers as a potential adjunct in root canal treatment by removing the smear layer before filling the root canal system, enhancing the adhesion of sealers to dentin and improving the sealing ability. AIM: To evaluate the microtensile bond strength of AH-Plus resin-based sealer to dentin after treatment with 980 nm diode and 1,064 nm neodymium-doped:yttrium aluminum garnet (Nd:YAG) laser in vitro. MATERIALS AND METHODS: Thirty specimens prepared for three groups namely group I (control), group II (980 nm diode-lased specimens) and group III (Nd:YAG-lased specimens). One tooth from each group was observed under scanning electron microscope for evaluation of intracanal root dentin morphology. Remaining specimens were used for making microsections by hard tissue microtome. Specimens for groups II and III were lased with 980 nm diode and 1,064 nm Nd:YAG laser. AH Plus sealer was applied onto specimens and mounted onto Instron universal testing machine for microtensile bond strength testing. Results were subjected to statistical analysis using one-way analysis of variance (ANOVA) and Tukey's test. RESULTS: Group III Nd:YAG had maximum mean microtensile bond strength values (11.558 ± 0.869), followed by group II diode (9.073 ± 0.468) and group I control (6.05 ± 0.036). Statistically significant differences were seen among all the groups. SEM analysis shows removal of smear layer in both groups II and III. CONCLUSION: Both Nd:YAG and diode laser were more effective than control group in improving the microtensile bond strength of AH Plus sealer to dentin. CLINICAL SIGNIFICANCE: Lasers have the potential to increase the adhesiveness of root canal sealer to dentin surface, thereby improving the quality of root canal obturation.
Asunto(s)
Recubrimiento Dental Adhesivo , Resinas Epoxi/química , Láseres de Semiconductores/uso terapéutico , Láseres de Estado Sólido/uso terapéutico , Materiales de Obturación del Conducto Radicular/química , Análisis del Estrés Dental/instrumentación , Dentina/cirugía , Dentina/ultraestructura , Humanos , Terapia por Láser/métodos , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Microtomía , Preparación del Conducto Radicular/instrumentación , Preparación del Conducto Radicular/métodos , Capa de Barro Dentinario , Estrés Mecánico , Resistencia a la TracciónRESUMEN
Recently developed, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive modality for mediastinal lymph node staging in lung cancer patients as well as for the diagnosis of mediastinal and hilar lymphadenopathy. It has been shown in systematic reviews and meta-analysis that a high diagnostic yield can be achieved with EBUS-TBNA for staging lung cancer. Though still not a standard of practice, this novel technology has attracted physicians and surgeons as an alternative modality to surgical biopsy for the assessment of the mediastinum. Standard cytology, thin layer preparations in liquid medium or cell blocks of cells obtained by EBUS-TBNA can be applicable not only for pathological diagnosis but also for further investigations such as immunohistochemistry and fluorescence in situ hybridization. In addition, samples obtained by EBUS-TBNA can also be used for molecular analysis. The key to a successful EBUS-TBNA is to understand the anatomy of the mediastinum as well as the basic steps of the procedure. Moreover, handling of the sample obtained by EBUS-TBNA is crucial for diagnosis since no amount of skill or interest of the interpreter can make up for a badly prepared sample. The goals of rapid on-site evaluation during EBUS-TBNA include determination of whether sampling of the target has been achieved and more importantly triage of samples to secondary investigations. This manuscript explains the detailed techniques of EBUS-TBNA to master this innovative procedure.
Asunto(s)
Biopsia con Aguja Fina/métodos , Bronquios/patología , Broncoscopía/métodos , Endosonografía/métodos , Ganglios Linfáticos/patología , Ultrasonografía Intervencional/métodos , Anestesia Local , Broncoscopios , Carcinoma de Pulmón de Células no Pequeñas/patología , Centrifugación , Sedación Consciente , Endosonografía/instrumentación , Diseño de Equipo , Granuloma/diagnóstico , Granuloma/patología , Humanos , Enfermedades Pulmonares/diagnóstico , Enfermedades Pulmonares/patología , Neoplasias Pulmonares/patología , Metástasis Linfática/diagnóstico , Linfoma/diagnóstico , Linfoma/patología , Mediastino , Microtomía , Estadificación de Neoplasias , Adhesión en Parafina , Manejo de Especímenes/métodos , Coloración y Etiquetado , Factores de Tiempo , Ultrasonografía Intervencional/instrumentaciónRESUMEN
Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca(2+) play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites. New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca(2+) responses in GFP-tagged neurons, a non-green fluorescent Ca(2+) indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca(2+) indicator fura-2 has been used in combination with GFP-tagged neurons. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals. Therefore, we decided to use a red fluorescent Ca(2+) indicator dye. The huge Stokes [corrected] shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons. Fura-red based Ca(2+) imaging was also successfully combined with GFP-tagged pancreatic ß-cells and GFP-tagged receptors expressed in HEK cells. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium. Therefore, the fluorescence of resting neurons with low Ca(2+) concentration has relatively high intensity. It should be noted, that other red Ca(2+)-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.
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Encéfalo/citología , Calcio/análisis , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Hipotálamo/citología , Microscopía Fluorescente/métodos , Neuronas/química , Animales , Encéfalo/metabolismo , Química Encefálica , Calcio/metabolismo , Hipotálamo/química , Hipotálamo/metabolismo , Ratones , Ratones Transgénicos , Microtomía/métodos , Neuronas/citología , Neuronas/metabolismo , Receptores LHRH/metabolismoRESUMEN
BACKGROUND AND AIMS: Cutting plant material is essential for observing internal structures and may be difficult for various reasons. Most fixation agents such as aldehydes, as well as embedding resins, do not allow subsequent use of fluorescent staining and make material too soft to make good-quality hand-sections. Moreover, cutting thin roots can be very difficult and time consuming. A new, fast and effective method to provide good-quality sections and fluorescent staining of fresh or fixed root samples, including those of very thin roots (such as Arabidopsis or Noccaea), is described here. METHODS: To overcome the above-mentioned difficulties the following procedure is proposed: fixation in methanol (when fresh material cannot be used) followed by en bloc staining with toluidine blue, embedding in 6 % agarose, preparation of free-hand sections of embedded material, staining with fluorescent dye, and observation in a microscope under UV light. KEY RESULTS: Despite eventual slight deformation of primary cell walls (depending on the species and root developmental stage), this method allows effective observation of different structures such as ontogenetic changes of cells along the root axis, e.g. development of xylem elements, deposition of Casparian bands and suberin lamellae in endodermis or exodermis or peri-endodermal thickenings in Noccaea roots. CONCLUSIONS: This method provides good-quality sections and allows relatively rapid detection of cell-wall modifications. Also important is the possibility of using this method for free-hand cutting of extremely thin roots such as those of Arabidopsis.
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Pared Celular/ultraestructura , Microscopía Fluorescente/métodos , Microtomía/métodos , Raíces de Plantas/citología , Coloración y Etiquetado/métodos , Arabidopsis/citología , Brassica napus/citología , Colorantes Fluorescentes , Cebollas/citología , Manejo de Especímenes , Triticum/citología , Zea mays/citologíaRESUMEN
Virotherapy offers a new treatment strategy using oncolytic viruses as self-replicating, tumor-specific agents, which destroy tumors during their natural lytic replication process. To study potential oncolytic viruses, cell culture experiments give basic information about the lytic potential of a virus, measured as cell lysis or decreased viability. For further analysis, animal models are usually employed. As these in vivo experiments are often performed in immunocompromised animals, results have to be interpreted with caution. Therefore, to obtain deeper information of the oncolytic action of specific viruses in a patient's individual context we established a test platform based on human primary tissue slices. In this three-dimensional model, we observed a preferential tumor infection and the penetration of oncolytic measles vaccine virus into deeper cell layers of tumor tissues, which is an essential feature of an effective oncolytic virus.
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Neoplasias Hepáticas/terapia , Viroterapia Oncolítica , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Genes Reporteros , Terapia Genética , Vectores Genéticos/genética , Células Hep G2 , Humanos , Microtomía/métodos , Virus Oncolíticos/genética , Células Tumorales Cultivadas , Virus Vaccinia/genéticaRESUMEN
Plants rich in condensed tannins are an alternative to chemical anthelmintics to control gastrointestinal nematodes (GINs) in ruminants. Previous functional studies have shown that sainfoin extracts affect the two forms of infective larvae (L3), ensheathed and exsheathed. However, the mechanisms of action remain unknown. The aim of this study was thus to compare ultrastructural changes in ensheathed and exsheathed L3 of two GIN species after in vitro contact with sainfoin extracts using transmission electron microscopy. The main changes identified were an alteration of the hypodermis, the presence of numerous vesicles in the cytoplasm and degeneration and/or death of muscular and intestinal cells. The changes suggested similar and nonspecies-specific lesions in the two nematode species. Comparison of the modifications found in the ensheathed vs. exsheathed L3s revealed different locations of the main cellular changes depending on the larval form. It is hypothesized that these spatial differences in lesions are mainly influenced by the presence of the sheath which favors contact between the active compounds and either the cuticle or the digestive tract. Overall, our observations suggest that the functional changes observed in the biology of GIN L3s after contact with sainfoin extracts are mediated through a direct mode of action, i.e. different interactions between the bioactive plant metabolites and the nematode structure depending on the route of contact.
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Antihelmínticos/farmacología , Fabaceae/química , Larva , Nematodos , Extractos Vegetales/farmacología , Rumiantes/parasitología , Animales , Antihelmínticos/química , Larva/efectos de los fármacos , Larva/ultraestructura , Microscopía Electrónica de Transmisión , Microtomía , Nematodos/efectos de los fármacos , Nematodos/ultraestructura , Extractos Vegetales/química , Adhesión en Plástico , Manejo de Especímenes , Taninos/química , Taninos/farmacologíaRESUMEN
Stress and obesity are highly prevalent conditions, and the mechanisms through which stress affects food intake are complex. In the present study, stress-induced activation in neuropeptide systems controlling ingestive behavior was determined. Adult male rats were exposed to acute (30 min/d × 1 d) or repeated (30 min/d × 14 d) restraint stress, followed by transcardial perfusion 2 h after the termination of the stress exposure. Brain tissues were harvested, and 30 µm sections through the hypothalamus were immunohistochemically stained for Fos protein, which was then co-localized within neurons staining positively for the type 4 melanocortin receptor (MC4R), the glucagon-like peptide-1 receptor (GLP1R), or agouti-related peptide (AgRP). Cell counts were performed in the paraventricular (PVH), arcuate (ARC) and ventromedial (VMH) hypothalamic nuclei and in the lateral hypothalamic area (LHA). Fos was significantly increased in all regions except the VMH in acutely stressed rats, and habituated with repeated stress exposure, consistent with previous studies. In the ARC, repeated stress reduced MC4R cell activation while acute restraint decreased activation in GLP1R neurons. Both patterns of stress exposure reduced the number of AgRP-expressing cells that also expressed Fos in the ARC. Acute stress decreased Fos-GLP1R expression in the LHA, while repeated restraint increased the number of Fos-AgRP neurons in this region. The overall profile of orexigenic signaling in the brain is thus enhanced by acute and repeated restraint stress, with repeated stress leading to further increases in signaling, in a region-specific manner. Stress-induced modifications to feeding behavior appear to depend on both the duration of stress exposure and regional activation in the brain. These results suggest that food intake may be increased as a consequence of stress, and may play a role in obesity and other stress-associated metabolic disorders.
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Proteína Relacionada con Agouti/metabolismo , Hipotálamo/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptor de Melanocortina Tipo 4/metabolismo , Receptores de Glucagón/metabolismo , Estrés Fisiológico , Proteína Relacionada con Agouti/genética , Animales , Apetito , Recuento de Células , Conducta Alimentaria , Expresión Génica , Receptor del Péptido 1 Similar al Glucagón , Habituación Psicofisiológica , Corazón , Hipotálamo/ultraestructura , Inmunohistoquímica , Masculino , Microtomía , Neuronas/metabolismo , Neuronas/ultraestructura , Obesidad/metabolismo , Perfusión , Proteínas Proto-Oncogénicas c-fos/genética , Ratas , Receptor de Melanocortina Tipo 4/genética , Receptores de Glucagón/genética , Restricción Física/fisiología , Transducción de Señal/fisiologíaRESUMEN
Understanding how specific proteins are degraded by neurons in living animals is a fundamental question with relevance to many neurodegenerative diseases. Dysfunction in the ubiquitin-proteasome system (UPS) specifically has been implicated in several important neurodegenerative diseases including Alzheimer's Disease, Parkinson's Disease, and amyotrophic lateral sclerosis. Research in this area has been limited by the fact that many inhibitors of the UPS given systemically do not cross the blood-brain barrier (BBB) in appreciable levels. This limits the ability to easily test in vivo specific hypotheses generated in reduced systems, like brain slice or dissociated cell culture, about whether the UPS may degrade a particular protein of interest. Although several techniques including intracerebral application via direct syringe injection, catheter-pump systems and drug-eluting beads are available to introduce BBB-impermeant drugs into brain they each have certain limitations and new approaches could provide further insights into this problem. In order to test the role of the UPS in protein degradation in vivo we have developed a strategy to treat mouse cortex with the UPS inhibitor clasto-lactacystin beta-lactone (CLBL) via a "cranial window" and recover the treated tissue for immunoblot analysis. This approach can be used in several different cranial window configurations including single window and double hemi-window arrangements that are tailored for different applications. We have also developed two different strategies for recovering treated cortical tissue including a vibratome/laser capture microscopy (LCM)-based and a vibratome only-based approach, each with its own specific advantages. We have documented UPS inhibition >600µm deep into the cortex with this strategy. This set of techniques in the living mammalian brain is complementary to previously developed approaches and extends the repertoire of tools that can be used to the study protein degradation pathways relevant to neurodegenerative disease.