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

Adulti (3-4 mesi) topi maschi su un misto C57BL / 6 di fondo sono stati utilizzati in questo protocollo. Gli animali sono stati tenuti al 12 hr luce / buio ciclo, e consentito l&#39;accesso gratuito al cibo e all&#39;acqua. Tutte le procedure eseguite in questo protocollo sono stati condotti secondo protocolli approvati dalla University Istituzionale cura e l&#39;uso degli animali Comitato Drexel. 1. Preparazione Area Chirurgica <ol><li> Disinfettare tavolo operatorio con il 70% di etanolo, quindi coprire l&#39;intera panchina chirurgica con assorbenti e organizzare strumenti chirurgici adiacente stereotassica. </li><li> Impostare attrezzature stereotassico senza braccio manipolatore. Disporre il rilievo di riscaldamento sul stereotassica e impostato a 37 ° C. Evitare il surriscaldamento l&#39;animale mettendo un piccolo pezzo di carta assorbente o tampone chirurgica tra l&#39;animale e il rilievo di riscaldamento. </li><li> Utilizzando le forbici in autoclave, tagliare piccoli pezzi di Gelfoam autoclavato in una piastra di Petri sterile contenente soluzione fisiologica sterile allo 0,9% fino a ready all&#39;uso. Nota: Mantenere ambiente di lavoro sterile, utilizzando strumenti in autoclave e rifornimenti chirurgici sterili. Re-sterilizzare gli strumenti chirurgici durante la procedura o in tra gli animali mediante immersione in uno sterilizzatore tallone per 10-15 secondi, a seconda delle necessità. Mantenere guanti puliti durante l&#39;intera procedura sfregando le mani con il 70% di etanolo, se necessario, da disinfettare. </li></ol> 2. Prepping mouse per Chirurgia <ol><li> Rimuovere mouse dalla gabbia di casa e pesare (g). </li><li> Posizionare il mouse nella camera di induzione isoflurano e ossigeno impostata su 2 L / min e vaporizzatore isoflurano a 5 per indurre un piano chirurgico di anestesia, circa 3-5 minuti. Monitor per la respirazione rallentata e l&#39;immobilizzazione. Verificare che il mouse sia completamente sedato con la reflex pizzico piedi. </li><li> Quando il mouse è completamente sedato, metterli in telaio stereotassico, fissare il naso nel cono naso, che è collegato con un tubo alla isoflurano. Inserire barre di orecchio in canale uditivo e stringere, garantendo la testa è stabile. </li><li> Radere la testa da un orecchio all&#39;altro, e da mezzo agli occhi di dietro le orecchie. </li><li> Sterilizzare la pelle con alternanza di salviette di alcol isopropilico e soluzione betadine iodio, 3 volte ciascuno. </li><li> Applicare lacrime artificiali per entrambi gli occhi per evitare che si secchi durante la procedura chirurgica. </li></ol> 3. Procedura chirurgica <ol><li> Monitorare la profondità dell&#39;anestesia pizzicando la punta o la coda. Il topo è nel piano chirurgica appropriata quando non vi è alcuna risposta, e la respirazione è lenta e uniforme. </li><li> Effettuare una incisione cutanea parasagittale da appena dietro gli occhi per quasi tra le orecchie in un unico movimento ferma con una lama n ° 11 bisturi. Spostare la pelle da parte e la clip a destra con pinza emostatica. </li><li> Cancella cranio di membrana sovrastante con il lato opaco del bisturi n ° 11 e il cotone punta applicatori. Facoltativamente, pulire il cranio con cotone applicatore capovolto immerso in soluzione salina allo 0,9%. Lasciare asciugare completamente. </li><li> Noiing un piccolo righello, segnare il bordo anteriore del craniotomia a 1 millimetro caudale alla sutura coronale, e il bordo sinistro del craniotomia a 1 mm lateralmente alla sutura sagittale (figura 1), con un pennarello indelebile. Quindi contrassegnare il bordo destro e caudale della craniotomia a 4 mm dal suture sagittali e coronali, rispettivamente (Figura 1). </li><li> Utilizzando una punta 0,5 millimetri, cominciare a fare craniotomia perforando lentamente seguendo il marcatore contorno permanente. Fare attenzione a non sfondare il cranio completamente. Premere delicatamente sul pezzo isolato di osso parietale con n ° 5 pinze, aree di debolezza darà modo alla pressione. Quando l&#39;osso assottigliata è sufficientemente debole per tutto il perimetro, il pezzo di osso è pronto per la rimozione. NOTA: Se la difficoltà esperienze investigatore rimuovendo l&#39;osso in un unico pezzo, questo suggerisce che l&#39;osso non era sufficientemente diluito durante la perforazione. Si consideri la foratura ulteriormente il cranio nelle successive animali al facilitafacile rimozione te del pezzo dell&#39;osso. </li><li> Usando una siringa da 10 ml munito di ago G 23, applicare una piccola quantità di soluzione fisiologica 0,9% per assorbire l&#39;osso isolata e zona forata. </li><li> Fissare il braccio manipolatore all&#39;apparecchiatura stereotassica. Collegare un nuovo n ° 11 bisturi al titolare sonda con il lato tagliente della lama rivolta verso rostralmente. NOTA: Sebbene i tessuti rostrale e caudale sperimentano gli spigoli smussati e della lama, rispettivamente, il danno meccanico indotto dalla ferita penetrante è comparabile in tutta l&#39;estensione della lesione. Osserviamo differenze apprezzabili caratteristiche principali di gliosi reattiva tra cui upregulation di espressione GFAP o la proliferazione, tra le sezioni rostrali e caudale. </li><li> Tenendo il braccio manipolatore di mezzo, sollevare con cautela l&#39;osso isolato con 5/45 pinze ad angolo. Inserire la punta della pinza nella parte dell&#39;osso isolata e ascensore, utilizzando la leva per tirare fuori il pezzo di osso lasciato in uno fulil movimento l. NOTA: Fare attenzione a non colpire il cervello o turbare la dura sotto il cranio. </li><li> Prendete un piccolo pezzo di schiuma gel assorbibile imbevuto e posizionare sul cervello scoperto per evitare che si secchi e assorbire il sangue che potrebbero essere presenti. </li><li> Una volta che la schiuma di gel è a posto, oscillare il braccio manipolatore in posizione e regolare lama centro craniotomia sopra la schiuma gel. Rimuovere la schiuma gel e abbassare la lama fino a quando la punta tocca la dura, senza pungere la dura. Mark dorsale / ventrale coordinate utilizzando il nonio sul braccio verticale della stereotassico. </li><li> Utilizzando il braccio manipolatore, abbassare lentamente la lama precisamente da 3 mm nel cervello. Ciò si ottiene utilizzando le marcature nonio sul braccio manipolatore. Lasciare lama per rimanere sul posto per 5-10 secondi. Portare il braccio stereotassica con attacco lama rostrale a caudale tre volte permettendo la lama per raggiungere i confini rostrale e caudale della craniotomia prima di passare alla opposifine te. NOTA: La dura non viene rimosso prima di inserire la lama. In contrasto con la dura madre ratto, che è ~ 80 micron di spessore 7, la dura mouse è considerevolmente più sottile (spessore di pochi strati di cellule) e non produce una resistenza apprezzabile bisturi durante l&#39;inserimento. Utilizzare una nuova lama di bisturi per ogni mouse per garantire che ogni animale riceve una lesione coerente. </li><li> Lentamente sollevare il braccio stereotassico, rimuovere la lama dal cervello. Dopo la rimozione della lama, immediatamente posto un altro pezzo di Gelfoam sulla superficie del cervello per assorbire qualsiasi eccesso di sangue o fluido. </li><li> Nel frattempo, smontare il braccio stereotassica e smaltire la lama n ° 11 bisturi. Una volta emorragia si è fermata, rimuovere il Gelfoam. </li><li> Chiudere la ferita sutura la pelle con sutura non riassorbibile, come ad esempio Ethilon o prolene. Le suture devono essere rimossi 9-10 giorni dopo l&#39;intervento. </li><li> Ritorna il mouse per la sua gabbia di casa, e lasciare il mouse per recuperare lentamente su un heating pad e monitor per eventuali segni di sofferenza. Il risveglio dall&#39;anestesia isofluorane indotta si verifica in genere entro 2-5 minuti dopo la rimozione dal isofluorane. Non lasciare l&#39;animale incustodito fino a quando non ha riconquistato recumbancy sternale. </li><li> Somministrare 0,5-1 ml di soluzione di Ringer lattato per via sottocutanea per garantire idratazione. </li></ol> Cura 4. post-chirurgico <ol><li> Seguire da vicino gli animali dopo l&#39;intervento fino al recupero dall&#39;anestesia, prima di tornare alla colonia. <ol><li> Per ridurre il dolore e il disagio post-operatorio, amministrare 0,05-0,1 mg / kg buprenorfina per iniezione ip immediatamente dopo la procedura. </li><li> Osservare gli animali per 2-3 giorni dopo l&#39;intervento per gravi segni di sofferenza, come i movimenti limitati, la mancanza di cura, o la perdita di peso. Eutanasia animali che dimostrano uno qualsiasi di questi segni di sofferenza e rimuovere dallo studio. </li></ol></li><li> Per esaminare istopatologia dei tessuti lesi, eutanasia animali da stanDard perfusione intracardial. <ol><li> Brevemente, anestetizzare animali con un sovradosaggio di ketamina / xilazina, quindi intracardiaca perfuso con 15-20 ml di NaCl allo 0,9%, o fino a quando il fegato è liberata di sangue, seguiti da 60 ml di 4% paraformaldeide a conclusione dell&#39;esperimento. </li></ol></li><li> Sezionare cervelli e post-correzione per 2-4 ore in paraformaldeide al 4% prima del trasferimento a una soluzione di saccarosio al 30%. Cervelli sezione su un criostato a 40-60 micron, e il processo di procedure istologiche o immunoistochimiche standard, oppure come descritto in Garcia 8,9. </li></ol>

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The authors have nothing to disclose.

E &#39;fondamentale che il cranio o dura sottostante non vengano danneggiati durante la perforazione. Utilizzare una leggera pressione durante la perforazione al fine di garantire il cranio non è forato. Inoltre, si deve prestare attenzione mentre si solleva il pezzo del cranio per garantire la dura non è decollato con l&#39;osso. L&#39;infortunio prosencefalo pugnalata qui descritto modelli una ferita penetrante al SNC. Anche se meno clinicamente traducibile di modelli TBI come la FPI e C...

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.

Dato che gli animali sottoposti a questa procedura non richiedono periodi sopravvivenza specializzati cura post-operatorio, brevi o time-lungo termine sono facilmente incorporati nello studio, a seconda delle necessità di indagare patologie acute o croniche a seguito di infortunio. Caratteristiche principali di gliosi reattiva, come l&#39;up-regolazione di GFAP e l&#39;ipertrofia del soma, si può osservare come già 2-3 giorni dopo un trauma. La fase di picco della proliferazione di astrociti reattivi è nei giorni 3-...

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.

Attivazione reattiva Gliosi<em&gt; In Vivo</em&gt; Da un Forebrain Stab Injury

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 Views.  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. 

Video: 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.

Un In vivo Modello di roditore della contrazione indotta Lesioni e monitoraggio non invasivo di recupero

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

Ricerca

Medicina

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.

Impianto di un bracciale carotidea per innescare Shear-stress aterosclerosi indotta in topi

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 MRI per il tracciamento cellulare

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.

Telemetria intrauterina misurare mouse contrattile Pressione<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.

Rilevazione di Anastasis In Vivo di biosensore CaspaseTracker

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.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

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 ...

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.

Aprire tracheostomia gastrico acido aspirazione Modello murino di lesioni polmonari acute Risultati in massima acuta non letale 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.

Riproducibile arteriosa Denudation Lesioni da infrarenale aortico addominale di serraggio in un modello murino

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 modello di ferita Drosophila In Vivo per lo studio della neurorigenerazione la periferica e del sistema nervoso centrale

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

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Indurre lesioni polmonari acute nei topi mediante instillazione diretta del lipopolioaccaride intracheale

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...