<ol><li> Anestetizzare topi con isoflurano (4% per l&#39;induzione, 1,5-2% per la chirurgia) utilizzando IACUC procedure approvate. E &#39;importante che la coda e / o pizzichi punta sono utilizzati al fine di assicurare l&#39;animale è completamente sedato. </li><li> Utilizzando un trimmer roditore, radere i capelli dalla parte posteriore del collo fino agli occhi. </li><li> Posizionare il mouse in una cornice stereotassica, nel corso di un intervento chirurgico d&#39;acqua a ricircolo di coperta. Fissare saldamente la testa con le barre orecchio. </li><li> Applicare una pomata oculare, al fine di prevenire gli occhi dell&#39;animale si secchi. </li><li> Somministrare, per via sottocutanea, Desametasone (0,2 mg / Kg) e carprofen (5 mg / Kg) per prevenire il gonfiore del cervello e / o una risposta infiammatoria, rispettivamente. </li><li> Prima di iniziare l&#39;intervento, sterilizzare l&#39;area operativa strofinando la pelle con tre colpi alternati di alcool al 70% e Betadine. </li><li> Tutti gli strumenti chirurgici sono stati pre-sterilizzati con uno sterilizzatore perle di vetro. Utilizzando le forbici che sono stati sterilizzati con etanolo, togliere la pelle sopra la parte superiore del cranio, a partire da un taglio orizzontale lungo tutta la base della testa, seguito da due tagli nella direzione rostrale, raggiungendo quasi le palpebre, poi due tagli obliqui che convergono sulla linea mediana. </li><li> Una goccia di adrenalina lidocaina + soluzione è applicata a questo punto sul periostio per evitare un eccessivo sanguinamento o dolore. Con un bisturi, ritrarre il periostio ai bordi del cranio. Inoltre, leggermente ritirare la muscolatura della parte posteriore del collo. </li><li> Raschiare delicatamente tutta l&#39;area esposta del cranio con il bisturi per creare una superficie asciutta. Questo è molto importante, in quanto consentirà la colla per aderire meglio se applicato in seguito. </li><li> Una volta che un sito di imaging è stato scelto, si è pronti per creare la finestra del cranio. In primo luogo, gentilmente &quot;disegnare&quot; un cerchio di circa 4 mm di diametro con il martello pneumatico dentale. </li><li> Dopo una foratura leggero, applicare lidocaina + adrenalina soluzione ancora una volta sulla superficie del cranio. Interrompere la perforazione quando uno strato molto sottile di osso è rimasto. Premendo delicatamente sul centro della craniotomia per sentire come si dà modo, si può sapere che di solito questa fase viene raggiunto. </li><li> Sotto una goccia di soluzione salina e prendendo vantaggio delle trabecole ossee - la struttura spugnosa delle ossa - ascensore via la craniotomia dal cranio con una pinza punta molto sottile. La salina è importante, in quanto contribuirà a sollevare il cranio e prevenire il sanguinamento della dura. </li><li> Applicare Gelfoam che è stato precedentemente lavate con soluzione alla dura madre per fermare qualsiasi emorragia di piccole dimensioni che si verifica di tanto in tanto quando il cranio è stato rimosso. </li><li> Dopo l&#39;essiccazione la superficie dura madre e la garanzia che non c&#39;è sanguinamento, appoggiare delicatamente una sterile di vetro da 5 millimetri di copertura antiscivolo sulla parte superiore della dura madre. (Nota: altri gruppi anche mettere una goccia di agarosio a basso punto di fusione (1,2%) oltre la dura e mettere il coprioggetto in cima alla agar). </li><li> Applicare una goccia di cianoacrilato colla a base all&#39;emisfero opposto sul cranio. Con l&#39;aiuto di un ago, esercitare il collante di tutto il finestra mentre facendo attenzione a non metterla sotto il vetro. Colla può essere applicata in uno strato sottile su tutta la superficie del cranio. </li><li> Una volta che la colla si è asciugata, mescolare acrilico dentale e applicarla su tutta la superficie del cranio, che contempli anche un orlo piccolo della ricevuta di copertura, per fissarlo. </li><li> Dopo essersi assicurato la polizza di copertura, fare un piccolo pozzo intorno alla finestra con acrilico dentale. Inoltre, integrare una barra di titanio in acrilico dentale. Questa barra sarà in seguito utilizzato per collegare il mouse in modo sicuro sul palco del microscopio per l&#39;imaging. E &#39;importante assicurarsi che il bar è di livello, in modo che sia parallelo con la finestra del cranio. Mettendo un pezzo di carta sotto la barra può permettere la barra di restare livello mentre l&#39;acrilico si indurisce. </li><li> L&#39;acrilico dentale è permesso di curare (indurimento) per 10 minuti, quando ormai la barra di titanio è fissata al suo posto. Posto l&#39;animale in una gabbia di caldo fino a che non recupera. </li><li> Dopo il recupero dall&#39;anestesia, l&#39;animale può essere ripreso nello stesso giorno. </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>

Come abbiamo mostrato nel video e nelle figure supplementari, la preparazione window cranica, combinato con l&#39;uso della microscopia a due fotoni, è uno strumento molto potente per studiare in vivo la struttura e la funzione della neocorteccia. La tecnica richiede un addestramento rigoroso per acquisire familiarità con l&#39;anatomia e le relative procedure chirurgiche e sottile abilità che richiede questa preparazione. Solo interventi chirurgici incontaminate possono essere utilizzati per l&#39;imaging cronica. Se la durata è manipo...

a craniotomy surgery procedure for chronic brain imaging

Tecniche di imaging stanno diventando sempre più importante nella funzione del cervello di studio. Tra questi, la microscopia a due fotoni scansione laser è emerso come un metodo estremamente utile, perché permette lo studio del cervello vive intatto. Con i preparativi del caso, questa tecnica permette l&#39;osservazione della stessa area corticale cronica, da minuti a mesi. In questo video ci mostra una preparazione cronica imaging in vivo del cervello usando la microscopia a due fotoni. Questa tecnica è stata inizialmente introdotta da Dott. Karel Svoboda, che ora è un Howard Hughes Medical Institute presso Janelia Farm. Preparati come quella mostrata qui può essere utilizzata per l&#39;imaging della struttura neocorticale (ad esempio, dinamiche dendritica e assonale), per registrare l&#39;attività neuronale con calcio-sensibili coloranti, alle dinamiche del flusso sanguigno corticale immagine, o per intrinseca studi di imaging ottico. L&#39;imaging profondo della neocorteccia è possibile con interventi chirurgici ottimale finestra cranica. Che operano nelle condizioni più sterile possibile per evitare infezioni, insieme con estrema cura per non danneggiare la dura madre durante l&#39;intervento, si tradurrà in successo e di lunga durata con tetto in vetro finestre craniche.

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

Questo video e protocollo dimostrano come impiantare una copertura in vetro finestra cranica nei roditori. Queste preparazioni possono essere utilizzate per cronica in vivo a due fotoni di imaging della neocorteccia su scale temporali di mesi. Può essere utilizzato anche per altri tipi di imaging, tra cui l&#39;imaging ottico del segnale intrinseco.

Un Chirurgia Procedura Craniotomia per l&#39;imaging cerebrale cronica

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

63.7K Views.  University of California, Los Angeles. Questo video e protocollo dimostrano come impiantare una copertura in vetro finestra cranica nei roditori. Queste preparazioni possono essere utilizzate per cronica in vivo a due fotoni di imaging della neocorteccia su scale temporali di mesi. Può essere utilizzato anche per altri tipi di imaging, tra cui l'imaging ottico del segnale intrinseco.

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.

Applicazione della Luce cured resina adesiva dentale per montaggio elettrodi o sonde Microdialisi negli esperimenti cronica

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

Ricerca

Biologia

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-drive array per la cronica In vivo</emRegistrazione>: Drive Fabrication

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-drive array per la cronica In vivo</emRegistrazione>: tetrodo Assemblea

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.

Murino procedura trapianto renale

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.

Vivere Imaging of the Brain Zebrafish embrionali da microscopia confocale

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.

Fare Giocare MR Child Imaging - Protocollo Neuroimaging Pediatrica, linee guida e 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 ...

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.

Imaging bioluminescenza di eme ossigenasi-1 sovraregolazione alla Procedura di 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.

Mappatura delle postumi di Theta Burst stimolazione sulla corteccia uditiva umana con imaging funzionale

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.

L&#39;isolamento di epatociti umani da un Two-step Collagenase perfusione procedura

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

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Prelievo di sangue dalla vena della coda laterale del Topo

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Methods Article

Un Chirurgia Procedura Craniotomia per l'imaging cerebrale cronica