Nous tenons à remercier JW Lichtman pour ses conseils en observation in vivo de muscles.

Coloration de la membrane de RBC <ol><li> Cellules rouges du sang de souris ont été corrigées à partir de la souche de souris même avec héparinisation. </li><li> Souris RBC ont été colorés avec PKH26 colorant (551/567 nm; rouge kit fluorescentes linker cellulaire, Sigma, USA). </li><li> RBC ont été lavés dans du PBS et incubées avec une solution de 2 pm PKH26 colorant pendant 5 minutes à température ambiante. La réaction a été stoppée par l&#39;addition de plasma (inactivé par la chaleur pendant 1 heure à 65 ° C à l&#39;avance) et incubées pendant 1 minute. </li><li> Les vitraux de RBC ont été lavées 5 fois avec du PBS. </li></ol> Préparation de la souris <ol><li> Trois à six mois masculine C57BL/10 souris ont été utilisées dans cette étude. Nous souris anesthésiés par injection intrapéritonéale de pentobarbital (50mg/kgBW). </li><li> Souris ont ensuite été intubé avec un tube de polyéthylène 20-Gage, ventilé mécaniquement et chauffé à 37 ° C sur un chauffe-feuille de Kapton relié à un capteur à thermocouple et contrôleur de système rétroaction de la température. </li><li> La face ventrale du cou a été rasé. Les muscles sterno-droite et de gauche ont été exposés. Les muscles ont été soutenus par un support en acier inoxydable et perfusées avec stériles Krebs Ringer solution. </li></ol> Injection <ol><li> Globules rouges colorées ont été réchauffée à 37 ° C, dilué avec de Krebs Ringer. 50ul de vitraux RBC (hématocrite de 12%) a été injecté dans la souris dans la veine du pénis. </li></ol> Dans l&#39;observation microscopique in vivo des muscles squelettiques Nous avons suivi la méthode de Jeff W. Lichtman &#39;s avec modification 1: <ol><li> Les animaux ont été placés sur un coussin chauffant au-dessus d&#39;un stade de fer fait à la main d&#39;un Nikon Eclipse-800 microscope. Eau-trempage des lentilles d&#39;objectif ont été utilisés pour vivre épi-fluorescence observation. Nous avons observé l&#39;intérieur teinté PKH26 RBC écoulement du vasculaires sous la lumière fluorescente d&#39;une lampe au mercure. Nous avons identifié les artérioles primaire, secondaire, artérioles, capillaires et les veines de leurs directions d&#39;écoulement des globules rouges et les architectures. </li><li> Une caméra SIT intensifié (Hamamatsu Photonics, C2400, Japon) et la vidéo-frame grabber ont été installés pour capter le signal de faible intensité à la cadence vidéo en temps réel (30 images par seconde). Les images ont été enregistrées sur un DVD sous forme de fichiers vidéo. </li></ol> Stimulation musculaire <ol><li> Une impulsion électrique est généré en utilisant un stimulateur nerveux périphérique (Innervator 252, Fisher &amp; Paykel Healthcare, la Nouvelle-Zélande), et livrés à la surface musculaire via une sonde revêtement inoxydable. Une extrémité de la sonde minus a été placé sur la partie proximale du muscle et une fin plus a été placé sur la partie distale du muscle. Stimulations tétanos ont été donnés au taux de 50Hz pendant 5 secondes. Les stimuli électriques ne causent pas de dommages fibres musculaires direct sur le voisinage de la surface de contact. </li></ol> RBC flux de l&#39;analyse <ol><li> Enregistré images vidéo de DVD ont été transférés les fichiers vidéo via un logiciel d&#39;image Savant frame grabber (Video Savant, USA). Les images vidéo ont été examinés dans la vitesse ajustée afin de visualiser différents teinté RBC. RBC flux est mesuré par comptage de RBC, qui volait à travers une artériole concentré primaires par minute. </li></ol>

<ol>
	<li>Lichtman, J. W., Magrassi, L., Purves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+neuromuscular+junctions+over+periods+of+several+months+in+living+mice.">Visualization of neuromuscular junctions over periods of several months in living mice.</a> J Neurosci. 7, 1215-1222 (1987).</li></ol>

Toutes les procédures liées à l&#39;expérimentation animale ont été examinés et approuvés par le Sous-Comité de protection des animaux de recherche du Massachusetts General Hospital.

Parmi les points techinical sont comme suit: (1) maintien de l&#39;état physiologique de l&#39;animal (aération, pH de la solution de la température du corps perfusative,), le volume d&#39;injection (2) de l&#39;tachés de RBC, et (3) les conditions d&#39;observation (optique optimale sélection, l&#39;intensité de fluorescence). Applications potentielles futures sont les suivants: (a) combinaison avec pharmacophysiological et / ou moléculaires interventions biologiques, (b) observation à long terme des artério / artériolo-sclérose...

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		<th>Material Name</th>
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		<th>Comment</th>
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<td>PKH26 dye </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>551/567 nm; Red fluorescent cell linker kit</td>
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<td>C57BL/10 mice </td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Three to six month old males</td>
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<td>pentobarbital </td>
<td>&nbsp;</td>
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in vivo micro-circulation measurement in skeletal muscle by intra-vital microscopy

CONTEXTE: Les facteurs réglementaires et de la physiologie de la microcirculation détaillée des vivo sont restés pas totalement clarifiée après de nombreuses différentes modalités d&#39;imagerie avait inventé. Alors que de nombreux paramètres macroscopiques de l&#39;écoulement sanguin reflète la vitesse d&#39;écoulement, des changements dans la vitesse d&#39;écoulement du sang et de globules rouges (GR) de flux ne tient pas de relation linéaire dans les observations microscopiques. Il ya des rapports de divergence entre RBC et RBC vitesse de flux, de flux et le volume de RBC flux de plasma, et de l&#39;hétérogénéité spatiale et temporelle de la régulation du débit dans les tissus périphériques dans les observations microscopiques, une base scientifique pour l&#39;exigence d&#39;études plus détaillées en matière de réglementation microcirculatoire aide microscopie intravitale. Méthodes: Nous avons modifié la méthode de Jeff Lichtman de l&#39;observation microscopique in vivo dans des muscles sterno-souris. Les souris sont anesthésiées, aéré, et injecté avec PKH26L-fluorescence globules rouges marqués pour l&#39;observation microscopique. RESULTAT ET CONCLUSIONS: globules rouges marqués par fluorescence sont détectées et distinguées ainsi par un microscope à champ large. La contraction musculaire provoquée par augmentation de la stimulation électrique induite dans le flux de RBC. Quantification des autres paramètres, y compris la vitesse de RBC et de la densité capillaire étaient réalisables. Souris bien toléré la chirurgie, injection de globules rouges colorés, l&#39;observation microscopique, et la stimulation électrique. Aucun muscle ou endommager les vaisseaux sanguins a été observée, suggérant que notre méthode est relativement moins invasive et adaptée aux observations à long terme.

Watch this Scientific Journal Video about In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy at JoVE.com

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

Une nouvelle méthode polyvalente pour l&#39;observation de la microcirculation est présenté. Il est considéré comme convenable pour observation à long terme, et pour la combinaison avec pharmacophysiological ou moléculaire interventions biologiques.

In vivo la micro-circulation de mesure dans le muscle squelettique par voie intra-vitale microscopie

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

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Research

JoVE Journal

Biology

11.1K vues.  Massachusetts General Hospital, and Harvard Medical School. Une nouvelle méthode polyvalente pour l'observation de la microcirculation est présenté. Il est considéré comme convenable pour observation à long terme, et pour la combinaison avec pharmacophysiological ou moléculaire interventions biologiques.

Vidéo: In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Suivi dynamique de prise de greffe musculaire chez les petits animaux par des In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Recherche

Biologie

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basées sur la fluorescence de mesure de l&#39;entrée du calcium magasin fonctionnant dans des cellules vivantes: à partir de cellules cancéreuses en culture de fibre musculaire squelettique

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

Évaluation ex vivo de la contractilité, fatigabilité et isolées du Alternans muscles squelettiques

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Évaluation de calcium Sparks dans intactes les fibres musculaires squelettiques

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo suivi clonale de cellules souches hématopoïétiques et progénitrices Marqué par cinq protéines fluorescentes confocale et multiphotonique Microscopie

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

Mesure de la dynamique des microtubules par la microscopie du disque filature dans des spindles mitotiques monopolaires

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Mesure de l’absorption de glucose stimulée par l’insuline et la contraction dans le muscle squelettique mature isolé et incubé de souris

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Mesure in vivo de la fonction musculaire extensrice du genou chez la souris

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

Quantification de l’infiltration graisseuse des muscles squelettiques par décellularisation

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Pleins feux sur l’auteur : Quantification de l’infiltration graisseuse des muscles squelettiques basée sur la décellularisation  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.