<ol><li> Anestesiar a los ratones con isoflurano (4% para la inducción, 1,5-2% para la cirugía) con IACUC los procedimientos aprobados. Es importante que la cola y / o aprieta los pies se utilizan con el fin de garantizar que el animal está completamente sedado. </li><li> Uso de un optimizador de roedores, se afeitan el pelo de la parte posterior del cuello hasta los ojos. </li><li> Coloque el ratón en un marco estereotáxico, más de una cirugía de agua de recirculación de manta. Sujetar firmemente la cabeza con barras de oído. </li><li> Aplicar pomada para los ojos, con el fin de evitar que el ojo del animal que se sequen. </li><li> Administrar por vía subcutánea, dexametasona (0,2 mg / Kg) y carprofeno (5 mg / Kg) para prevenir la inflamación del cerebro y / o una respuesta inflamatoria, respectivamente. </li><li> Antes de comenzar la cirugía, la esterilización de la zona de trabajo limpiando la piel con tres golpes alternados de 70% de alcohol y betadine. </li><li> Todos los instrumentos quirúrgicos se han pre-esterilizados utilizando un esterilizador de cuentas de vidrio. Con la tijera que han sido esterilizadas con etanol, quitar la piel sobre la parte superior del cráneo, a partir de un corte horizontal a lo largo de la base de la cabeza, seguido por dos cortes en la dirección rostral, casi llegando a los párpados, luego dos cortes oblicuos que convergen en la línea media. </li><li> Una gota de solución de lidocaína con adrenalina + se aplica en este punto en el periostio para evitar el sangrado excesivo o dolor. Con un bisturí, retirar el periostio de los bordes del cráneo. Además, la ligera retracción de la musculatura de la parte posterior del cuello. </li><li> Raspe suavemente toda la superficie expuesta del cráneo con el bisturí para crear una superficie seca. Esto es muy importante, ya que permitirá que el pegamento se adhiera mejor cuando se aplica más tarde. </li><li> Una vez que un sitio de imágenes ha sido elegido, uno está listo para crear la ventana del cráneo. En primer lugar, con suavidad &quot;dibujar&quot; un círculo de unos 4 mm de diámetro con el taladro neumático dental. </li><li> Después de una ligera perforación, aplicar lidocaína solución de epinefrina + nuevo en la superficie del cráneo. Detener la perforación, cuando una capa muy delgada de hueso que queda. Presionando suavemente sobre el centro de la craneotomía para sentir cómo se le da forma, por lo general se puede saber que se alcanza esta etapa. </li><li> En una gota de solución salina y las ventajas de tomar de las trabéculas óseas - la estructura esponjosa del hueso - elevación de distancia de la craneotomía en el cráneo con unas pinzas punta muy fina. La solución salina es importante, ya que ayudará a levantar la cabeza y evitar el sangrado de la duramadre. </li><li> Aplicar Gelfoam que ha sido previamente lavadas con solución salina a la duramadre para detener cualquier sangrado pequeño que a veces ocurre cuando el cráneo es removido. </li><li> Después de secar la superficie de la duramadre y la garantía de que no hay sangrado, mucho cuidado, coloque un recipiente estéril de vidrio de 5 mm hoja de la cubierta en la parte superior de la duramadre. (Nota: otros grupos también se coloca una gota de bajo punto de fusión de agarosa (1,2%) a lo largo de la dura y poner el cubreobjetos en la parte superior del agar). </li><li> Aplique una gota de pegamento a base de cianoacrilato para el hemisferio opuesto en el cráneo. Con la ayuda de una aguja, aplique el pegamento alrededor de la ventana, mientras que teniendo cuidado de no ponerlo en el vaso. Pegue ahora se puede aplicar en una capa fina sobre toda la superficie del cráneo. </li><li> Una vez que el pegamento se haya secado, mezcla de acrílico dental y aplicarlo en toda la superficie del cráneo, que abarca también un pequeño borde de la hoja de la cubierta, para asegurarla. </li><li> Después de obtener la hoja de la cubierta, hacer un pequeño pozo alrededor de la ventana con acrílico dental. Además, integrar una barra de titanio en el acrílico dental. Esta barra se utilizarán posteriormente para conectar el ratón con seguridad en la platina del microscopio para obtener imágenes. Es importante asegurarse de que el bar está a nivel, de modo que sea paralelo a la ventana del cráneo. Colocar una hoja de papel debajo de la barra se puede permitir que la barra de permanecer nivel, mientras que el acrílico se endurezca. </li><li> El acrílico dental se deja curar (endurecer) durante 10 minutos, momento en que la barra de titanio se fija en su lugar. Colocar el animal en una jaula caliente hasta que se recupere. </li><li> Después de la recuperación de la anestesia, el animal se pueden obtener imágenes en el mismo día. </li></ol>

<ol>
	<li>Svoboda, K., Denk, W., Kleinfeld, D., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+vivo+dendritic+calcium+dynamics+in+neocortical+pyramidal+neurons."> In vivo dendritic calcium dynamics in neocortical pyramidal neurons.</a> Nature. 85, 161-165 (1997).</li><li>Lendvai, B., Stern, E. A., Chen, B., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+plasticity+of+dendritic+spines+in+the+developing+rat+barrel+cortex+in+vivo.">Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo.</a> Nature. 404, 876-881 (2000).</li><li>Trachtenberg, J. T., Chen, B., Knott, G. W., Feng, G., Sanes, J. R., Welker, E., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420, 788-794 (2002).</li><li>Portera-Cailliau, C., Weimer, R. M., DePaola, V., Caroni, P., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+modes+of+axon+elaboration+in+the+developing+neocortex.">Diverse modes of axon elaboration in the developing neocortex.</a> PLoS Biol. 3, (2005).</li><li>Holtmaat, A., Wilbrecht, L., Knott, G. W., Welker, E., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+and+cell-type-specific+spine+growth+in+the+neocortex.">Experience-dependent and cell-type-specific spine growth in the neocortex.</a> Nature. 441, 979-983 (2006).</li></ol>

Como hemos mostrado en el video y en las figuras complementarias, la preparación de la ventana del cráneo, combinada con el uso de la microscopia de dos fotones, es una herramienta muy poderosa para estudiar in vivo la estructura y función del neocórtex. La técnica requiere de un riguroso entrenamiento para familiarizarse con la anatomía pertinente y los procedimientos quirúrgicos muy bien y las habilidades que requiere esta preparación. Sólo cirugías originales pueden ser utilizados para obtener imágenes de crónica. Si la durac...

a craniotomy surgery procedure for chronic brain imaging

Las técnicas de imagen son cada vez más importante en la función cerebral de estudio. Entre ellos, dos fotones microscopía de escaneo láser se ha convertido en un método muy útil, porque permite el estudio del cerebro intacto en vivo. Con los preparativos adecuados, esta técnica permite la observación de la zona cortical misma crónica, de minutos a meses. En este vídeo, se muestra una preparación para la crónica en imágenes in vivo del cerebro usando la microscopía de dos fotones. Esta técnica fue inicialmente promovida por el Dr. Karel Svoboda, quien es ahora un Howard Hughes Medical Institute en la Granja Janelia. Preparaciones como la que se muestra aquí se puede utilizar para obtener imágenes de la estructura neocortical (por ejemplo, la dinámica, dendríticas y axonal), para registrar la actividad neuronal con tintes sensibles al calcio, a la imagen de la dinámica del flujo sanguíneo cortical, o para los estudios de imagen óptica intrínseca. Imágenes profundas de la corteza cerebral es posible con óptima cirugías ventana craneal. Operando bajo las condiciones más estériles posible para evitar infecciones, junto con el uso de un cuidado extremo para no dañar la duramadre durante la cirugía, se traducirá en éxito y duradera cubierta de cristal y craneal.

Watch this Scientific Journal Video about A Craniotomy Surgery Procedure for Chronic Brain Imaging at JoVE.com

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Este video y el protocolo de demostrar cómo la implantación de una cubierta de cristal ventana craneal en los roedores. Estas preparaciones se pueden utilizar para la crónica en vivo de dos fotones de imágenes de la corteza cerebral en escalas de tiempo de meses. También puede ser utilizado para otros tipos de imágenes, incluyendo imágenes ópticas señal intrínseca.

Un procedimiento de cirugía craneotomía para imágenes cerebrales crónicas

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

a-craniotomy-surgery-procedure-for-chronic-brain-imaging

Research

JoVE Journal

Biology

63.6K vistas.  University of California, Los Angeles. Este video y el protocolo de demostrar cómo la implantación de una cubierta de cristal ventana craneal en los roedores. Estas preparaciones se pueden utilizar para la crónica en vivo de dos fotones de imágenes de la corteza cerebral en escalas de tiempo de meses. También puede ser utilizado para otros tipos de imágenes, incluyendo imágenes ópticas señal intrínseca.

Video: A Craniotomy Surgery Procedure for Chronic Brain Imaging

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

Aplicación de curado con luz Resina Adhesivo Dental para los electrodos de montaje o sondas de microdiálisis en experimentos crónica

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Investigación

Biología

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

Micro-array de unidades de crónicos In vivo Grabación: Unidad de Fabricación

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential amplitude of action potential signatures over the channels of a tetrode allows for the isolation of single-unit activity from multi-unit signals.  The ability to precisely control the stereotaxic location and depth of the tetrode is critical for studying coordinated neural activity across brain regions.  In combination with a micro-drive array, it is possible to achieve precise placement and stable control of many tetrodes over the course of days to weeks.  In this protocol, we demonstrate how to fabricate and condition tetrodes using basic tools and materials, install the tetrodes into a multi-drive tetrode array for chronic in-vivo recording in the rat, make ground wire connections to the micro-drive array, and attach a protective cone onto the micro-drive array in order to protect the tetrodes from physical contact with the environment.



Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

Micro-array de unidades de crónicos In vivo Grabación: Tetrodo Asamblea

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Procedimiento murino Trasplante Renal

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Imágenes en vivo del cerebro del embrión de pez cebra mediante microscopía confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12, behavioral approaches 13, 14,15, 16-18 and simulation 19, the use of mock scanner areas 20,21, basic relaxation 22 and a combination of these techniques 23 have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.

Making MR Imaging Child&#039;s Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children.

Hacer jugar MR Imaging Niño - Protocolo neuroimagen pediátrica, Directrices y Procedimientos

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

Bioluminiscencia de imágenes de hemo oxigenasa-1 Upregulation en el procedimiento de Gua Sha

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Mapeo de las secuelas de la theta ráfaga de estimulación sobre la corteza auditiva humana con imágenes funcionales

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

El aislamiento de hepatocitos humanos por una colagenasa perfusión Procedimiento de dos pasos

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

La investigación de las interacciones proteína-proteína en las células vivas utilizando la transferencia de energía de resonancia de bioluminiscencia

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...