We thank Katherine Clark for technical assistance. A.D.R.G. is funded in part by 5K01MH097957-03

Adulte (3-4 mois) sur des souris mâles un mixte C57BL / 6 fond ont été utilisés dans ce protocole. Les animaux ont été maintenus sur un cycle de 12 heures de lumière / obscurité, et ont permis l&#39;accès libre à la nourriture et de l&#39;eau. Toutes les procédures effectuées dans ce protocole ont été menées selon des protocoles approuvés par le Comité soin et l&#39;utilisation Drexel University animaux dans les institutions. 1. Préparation zone chirurgicale <ol><li> Désinfecter table chirurgicale avec 70% d&#39;éthanol, puis couvrir l&#39;ensemble du banc chirurgicale avec des tampons absorbants et d&#39;organiser les instruments chirurgicaux adjacent à stéréotaxique. </li><li> Mettre en place l&#39;équipement stéréotaxique sans bras manipulateur. Disposer le coussin chauffant sur la stéréotaxique et mettre à 37 ° C. Éviter la surchauffe de l&#39;animal en plaçant un petit morceau de serviette en papier ou un tampon chirurgical entre l&#39;animal et le coussin chauffant. </li><li> Avec des ciseaux autoclave, couper des petits morceaux de gelfoam autoclave dans une boîte de Pétri stérile contenant une solution saline à 0,9% stérile jusqu&#39;à ready à l&#39;emploi. Remarque: Maintenir un environnement de travail stérile en utilisant des instruments à l&#39;autoclave et des fournitures chirurgicales stériles. Re-stériliser des instruments chirurgicaux au cours de la procédure ou entre les animaux par trempage dans un stérilisateur à billes pendant 10 à 15 secondes, selon les besoins. Maintenir des gants propres tout au long de la procédure en frottant les mains avec de l&#39;éthanol 70%, au besoin, à désinfecter. </li></ol> 2. Prepping souris pour la chirurgie <ol><li> Retirer la souris de la maison cage et peser (g). </li><li> Placez la souris dans la chambre de l&#39;induction de l&#39;isoflurane et définir l&#39;oxygène à 2 L / min et du vaporisateur isoflurane à 5 pour induire un plan chirurgical d&#39;anesthésie, environ 3-5 min. Surveiller les ralentissement de la respiration et de l&#39;immobilisation. Vérifiez que la souris est entièrement sous sédation en utilisant le réflexe orteil de pincement. </li><li> Lorsque la souris est entièrement sous sédation, placer dans un cadre stéréotaxique, sécuriser le nez dans le cône de nez, qui est attaché avec des tubes à l&#39;isoflurane. Insérez barres d&#39;oreilles dans le canal auditif et serrer, assurant la tête est stable. </li><li> Raser la tête de l&#39;oreille à l&#39;oreille, et d&#39;entre les yeux derrière les oreilles. </li><li> Stériliser la peau avec des lingettes alternées de l&#39;alcool isopropylique et une solution d&#39;iode bétadine, trois fois chacun. </li><li> Appliquer des larmes artificielles pour les deux yeux pour les empêcher de se dessécher pendant la procédure chirurgicale. </li></ol> 3. Procédure chirurgicale <ol><li> Surveiller la profondeur de l&#39;anesthésie en pinçant l&#39;orteil ou la queue. La souris est dans le plan chirurgical approprié lorsqu&#39;il n&#39;y a pas de réponse, et la respiration est lente et uniforme. </li><li> Faire une incision de la peau parasagittale partir juste derrière les yeux à peu près entre les oreilles en un seul mouvement ferme à l&#39;aide d&#39;une lame de bistouri n ° 11. Déplacez la peau de côté et le clip côté droit avec hémostatique. </li><li> Effacer le crâne de la membrane recouvrant utilisant le côté mat de la n ° 11 scalpel et applicateurs coton-tige. Eventuellement, essuyez le crâne avec du coton-tige trempé dans une solution saline à 0,9%. Laisser sécher complètement. </li><li> Nousing une petite règle, marquer le bord antérieur de la craniotomie à 1 mm caudale à la suture coronale, et le bord gauche de la craniotomie à 1 mm latéral à la suture sagittale (Figure 1), avec un marqueur permanent. Ensuite, marquez les bords droit et caudales de la craniotomie à 4 mm des sutures sagittale et coronale, respectivement (figure 1). </li><li> Utiliser un foret de 0,5 mm, commencer à faire craniotomie en perçant lentement en suivant le contour de marqueur permanent. Soyez sûr de ne pas briser le crâne complètement. Appuyez doucement sur la pièce isolée de l&#39;os pariétal avec n ° 5 forceps, les zones de faiblesse cédera à la pression. Lorsque l&#39;os amincie est suffisamment faible tout au long du périmètre, le morceau d&#39;os est prêt à être retiré. NOTE: Si l&#39;expérience d&#39;enquêteur des difficultés à retirer l&#39;os dans une seule pièce, ce qui suggère que l&#39;os n&#39;a pas été suffisamment éclairci au cours du forage. Considérez le forage du crâne en outre chez les animaux ultérieures facilitaenlèvement facile te de la pièce d&#39;os. </li><li> Utiliser une seringue de 10 ml munie d&#39;une aiguille 23 G, appliquer une petite quantité de solution saline 0,9% à tremper l&#39;os isolé et zone percée. </li><li> Fixez le bras manipulateur à l&#39;équipement stéréotaxique. Fixez une nouvelle lame de bistouri n ° 11 à la porte de la sonde avec le côté tranchant de la lame face rostralement. Remarque: bien que les tissus rostrale et caudale expérience des arêtes vives et émoussée de la lame, respectivement, les dommages mécaniques induites par la blessure pénétrante est comparable tout au long de l&#39;étendue de la lésion. Nous observons aucune différence appréciable dans les grandes caractéristiques de gliose réactive, y compris la régulation positive de l&#39;expression de la GFAP ou la prolifération, entre les sections rostrale et caudale. </li><li> Garder le bras manipulateur de la route, soulevez délicatement l&#39;os isolé en utilisant une pince à angle 5/45. Insérez le bout de la pince dans le côté de l&#39;os et un ascenseur isolé, en utilisant l&#39;effet de levier pour retirer le morceau d&#39;os laissés dans un full mouvement. NOTE: Attention à ne pas poignarder le cerveau ou perturber la dure sous le crâne. </li><li> Prenez un petit morceau de la mousse de gel absorbable trempé et le placer sur le cerveau à découvert pour l&#39;empêcher de sécher et absorber le sang qui pourrait être présente. </li><li> Une fois la mousse de gel est en place, balancer le bras manipulateur en place et régler la lame au centre de la craniotomie sur la mousse de gel. Retirez la mousse de gel et abaisser la lame jusqu&#39;à ce que la pointe touche la dure sans perforation de la dure-mère. Mark dorsale / ventrale coordonnées en utilisant le vernier sur le bras vertical de la stéréotaxique. </li><li> Utilisation du bras manipulateur, abaissez lentement la lame précisément 3 mm dans le cerveau. Ceci est réalisé à l&#39;aide des graduations coulisse sur le bras de manipulateur. Laisser la lame de rester en place pendant 5-10 sec. Déplacez le bras stéréotaxique avec l&#39;attachement de lame rostrale à caudale trois fois la lame permettant d&#39;atteindre les limites rostrale et caudale de la craniotomie avant de passer à l &#39;oppositionte fin. REMARQUE: La dure-mère est pas éliminé avant l&#39;insertion de la lame. A la différence de la dure-mère chez le rat, qui est d&#39;environ 80 um d&#39;épaisseur 7, la dure-mère de la souris est considérablement plus mince (quelques couches de cellules d&#39;épaisseur) et ne produit pas une résistance appréciable à la lame de scalpel pendant l&#39;insertion. Utiliser une nouvelle lame de scalpel pour chaque souris pour que chaque animal reçoit une blessure cohérente. </li><li> Soulevez lentement le bras stéréotaxique, retirer la lame du cerveau. Après le retrait de la lame, placer immédiatement un autre morceau de gelfoam sur la surface du cerveau d&#39;absorber toute l&#39;excès de sang ou de liquide. </li><li> Pendant ce temps, retirez le bras stéréotaxique et de disposer de la lame de bistouri n ° 11. Une fois que le saignement a cessé, retirez le gelfoam. </li><li> Fermer la plaie par suture la peau avec une suture non résorbable, comme Ethilon ou prolène. Sutures doivent être retirés 9-10 jours après la chirurgie. </li><li> Retour de la souris à sa cage, et laissez la souris pour récupérer lentement sur une heatinpad g et moniteur pour des signes de détresse. Récupération de l&#39;anesthésie induite isofluorane-se produit généralement dans les 2-5 min après le retrait de la isofluorane. Ne pas laisser l&#39;animal sans surveillance jusqu&#39;à ce qu&#39;il ait retrouvé décubitus sternale. </li><li> Administrer 0,5-1 ml de lactate sous-cutanée de la solution de Ringer pour assurer une hydratation. </li></ol> 4. Soins post-opératoire <ol><li> Surveillez attentivement les animaux post-opératoire jusqu&#39;à la récupération de l&#39;anesthésie avant de retourner à la colonie. <ol><li> Pour réduire la douleur et l&#39;inconfort post-opératoire, administrer 0,05-0,1 mg / kg buprénorphine par injection ip immédiatement après la procédure. </li><li> Observez les animaux pendant 2-3 jours après l&#39;opération pour des signes graves de détresse tels que les mouvements limités, absence de toilettage, ou la perte de poids. Euthanasier des animaux qui présentaient une de ces signes de détresse et de retirer de l&#39;étude. </li></ol></li><li> Pour examiner l&#39;histopathologie des tissus blessés, euthanasier les animaux par stanperfusion intracardiaque dard. <ol><li> En bref, anesthésier les animaux avec une surdose de kétamine / xylazine, puis intracardiaque perfusé avec 15 à 20 ml de NaCl à 0,9%, ou jusqu&#39;à ce que le foie est effacé de sang, suivie de 60 ml de paraformaldehyde à 4% à la fin de l&#39;expérience. </li></ol></li><li> Disséquer le cerveau et post-correctif pour 2-4 heures dans 4% de paraformaldehyde avant de transférer à la solution de saccharose à 30%. Section cerveaux sur un cryostat à 40-60 um, et le processus de procédures histologiques ou immunohistochimiques standard, ou comme décrit dans Garcia 8,9. </li></ol>

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The authors have nothing to disclose.

Il est essentiel que le crâne ou de la dure sous-jacent ne sont pas endommagés pendant le forage. Utilisez une légère pression pendant le forage pour assurer le crâne est pas percé. En outre, il faut prendre soin tout en soulevant la pièce de crâne pour assurer la dure-mère n&#39;a pas décollé avec l&#39;os. La blessure du cerveau antérieur de couteau décrit ici des modèles d&#39;une blessure pénétrante au SNC. Bien que moins traduisible cliniquement que les modèles de TBI t...

A major challenge for developing successful therapies to treat the injured CNS is an incomplete understanding of the complex multicellular events that are triggered by the trauma. Reactive astrocytes are gaining increasing recognition as a promising target for novel therapies1. Though historically regarded as hostile to neural repair, reactive astrocytes are now recognized as critical components of a complex, multicellular neuroprotective response that includes attenuation of inflammatory processes and limiting secondary damage and neurodegeneration2-6. Although the neuropathological characteristics of reactive gliosis have long been well defined, the cellular and molecular mechanisms regulating reactive gliosis, and the diverse array of downstream consequences remain poorly understood. Understanding the mechanisms that drive reactive gliosis, as well as the subsequent cellular and molecular events, is an important step towards developing strategies aimed at promoting the neuroprotective properties of reactive gliosis, while attenuating the detrimental effects. 
Here we demonstrate a method to induce severe reactive astrogliosis in the forebrain of adult mice using a stab injury. In contrast to other traumatic brain injury (TBI) models, such as controlled cortical impact (CCI) or fluid percussion injury (FPI), which require specialized equipment to produce an injury, the forebrain stab requires only a stereotaxic device to stabilize the head and a No. 11 scalpel blade. Thus the forebrain stab lesion model is more broadly accessible to a wide range of laboratories that do not have access to the specialized devices necessary for creating an FPI or CCI injury. The method described here enables investigators to reliably and reproducibly trigger a robust gliosis response to investigate subsequent cellular and molecular events. Once recovered from surgery, animals that have received a forebrain stab injury can survive for prolonged periods without the need for specialized care and can be returned to the colony for acute, intermediate, or chronic studies. Though less clinically translatable than FPI or CCI models of TBI, a forebrain lesion produced by a stab injury serves as a simple yet useful experimental model to investigate basic biological mechanisms underlying reactive gliosis and other neuropathological events following trauma to the CNS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stereotax</td>
			<td>Harvard Apparatus</td>
			<td>726049</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed micro drill</td>
			<td>Harvard Apparatus</td>
			<td>724950</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel scalpel blade, #11</td>
			<td>MedVet</td>
			<td>JOR581S</td>
			<td></td>
		</tr>
		<tr>
			<td>5/45 angled forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam sponge 12cmx7mm</td>
			<td>Fisher</td>
			<td>NC9841478</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies and other reagents</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td>Dilution (brightfield) 
			Dilution (fluorescence)</td>
		</tr>
		<tr>
			<td>Rb anti-GFAP</td>
			<td>DAKO</td>
			<td>&#160;Z033429-2</td>
			<td>1:20k 
			1:1k</td>
		</tr>
		<tr>
			<td>Shp anti-BrdU</td>
			<td>Abcam</td>
			<td>ab1893</td>
			<td>1:2k 
			1:500</td>
		</tr>
		<tr>
			<td>Biotinylated goat anti-rabbit</td>
			<td>Vector Laboratories</td>
			<td>BA-1000&#160;</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Biotinylated rabbit anti-sheep</td>
			<td>Vector Laboratories</td>
			<td>BA-6000</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexafluor 488 goat anti-rabbit</td>
			<td>Life Technologies</td>
			<td>A-11008</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexafluor 568 donkey anti-sheep</td>
			<td>Life Technologies</td>
			<td>A-21099</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>DAPI Nucleic Acid Stain</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Cresyl Violet Acetate</td>
			<td>Sigma Aldrich</td>
			<td>C5042-10G</td>
			<td>1%</td>
		</tr>
	</tbody>
</table>

triggering reactive gliosis in vivo by a forebrain stab injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Parce que les animaux soumis à cette procédure ne nécessitent pas de périodes de survie soins post-opératoires spécialisés, à court ou à temps à long terme sont facilement incorporés dans l&#39;étude, en fonction de la nécessité d&#39;étudier la pathologie aiguë ou chronique suite à une blessure. Principales caractéristiques de gliose réactive, tels que la régulation positive de la GFAP et l&#39;hypertrophie du soma, peuvent être observés dès 2-3 jours après la blessure. La phase pic de la prolif...

Watch this Scientific Journal Video about Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury at JoVE.com

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Déclenchement réactive Gliose<em&gt; In Vivo</em&gt; Par un cerveau antérieur Stab blessures

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

triggering-reactive-gliosis-in-vivo-by-a-forebrain-stab-injury

Research

JoVE Journal

Medicine

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

10.4K vues.  Drexel University. This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods. 

Vidéo: Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Une In vivo</emModèle de rongeur> de contraction induite par des blessures et de surveillance non invasive de récupération

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Recherche

Médecine

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Implantation d&#39;une manchette carotidienne pour le déclenchement de cisaillement-stress athérosclérose induite chez la souris

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F IRM pour le suivi cellulaire

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Télémétrie intra-utérine mesurer souris contractile pression<em&gt; In Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Détection de Anastasis In Vivo par CaspaseTracker biocapteur

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Appauvrissement des cellules de souris de xénogreffes de tumeurs humaines améliore de manière significative l&#39;analyse en aval des cellules cibles

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Une blessure à la tête modèle Répétitive commotionnant chez la souris

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Ouvrir trachéotomie acide gastrique Aspiration modèle murin de lésion pulmonaire aiguë Résultats dans Maximal aiguë non létaux Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Reproductible Arterial dénudation blessures par infrarénale de l&#39;aorte abdominale de serrage dans un modèle murin

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

Un modèle de blessure de Drosophila In Vivo pour étudier la neurogenèse dans le périphérique et le système nerveux Central

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...