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

Karışık C57BL / 6 arka plan üzerine Yetişkin (3-4 aylık) erkek fareler bu protokol kullanıldı. Hayvanlar, 12 saat aydınlık / karanlık döngüsünde tutuldu ve gıda ve suya serbest olarak ulaşmalarına izin verilmiştir. Bu protokolde yapılan tüm işlemler Drexel Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylanan protokollere göre gerçekleştirilmiştir. 1. Cerrahi Alan hazırlanması <ol><li> % 70 etanol ile, cerrahi tablo dezenfekte, daha sonra, emici ped ile tüm cerrahi tezgah kapağı ve stereotaksik bitişik cerrahi aletler sağlayabilir. </li><li> Manipülatör kolunun olmadan stereotaksik ekipman ayarlayın. Stereotaksik ısıtma yastığı yerleştirin ve 37 ° C olarak ayarlayın. Hayvan ve ısıtma yastığı arasındaki kağıt havlu veya cerrahi ped küçük bir parça koyarak hayvan aşırı ısınma kaçının. </li><li> Otoklava makas kullanarak, Rea kadar% 0.9 steril tuzlu su çözeltisi içeren steril bir Petri kabı içine otoklava gelfoam küçük parçalarkullanım için dy. Not: otoklavlanmış aletleri ve steril cerrahi malzemeleri kullanarak steril bir çalışma ortamı sağlayın. Gerektiğinde, 10-15 saniye boyunca bir boncuk sterilizatör içine batırarak hayvanlar arasındaki işlem sırasında ya da cerrahi aletler yeniden sterilize edin. Dezenfekte etmek, gerektiği gibi,% 70 etanol ile ellerini ovuşturarak tarafından işlem boyunca temiz eldiven koruyun. </li></ol> Cerrahi 2. hazırlıyor Fare <ol><li> (G) ev kafesinden fareyi çıkarın ve tartın. </li><li> Izofluran indüksiyon odasına ve 5 2 L / dk ve izofluran buharlaştırıcı ayarlanmış oksijen içine yerleştirin fare anestezi cerrahi uçağı, yaklaşık 3-5 dakika ikna etmek. Yavaşlaması solunum ve immobilizasyon için izleyin. Fare tam ayak tutam refleks ile sedasyon olup olmadığını kontrol edin. </li><li> Fare tamamen uyuşturulmuş olduğunda, stereotaksik çerçeve içinde yer izofluran boru ile bağlı burun konisi, burun sabitleyin. Kafa sağlanması istikrarlı, kulak kanalı içine kulak çubukları yerleştirin ve sıkın. </li><li> Kulaktan kulağa tıraş ve gözler arasında gelen kulakların arkasında için. </li><li> Izopropil alkol ve betadin iyot çözeltisi, 3 kez her alternatif mendil ile cilt sterilize edin. </li><li> Cerrahi işlem sırasında kurumasını önlemek için her iki göze yapay gözyaşları uygulayın. </li></ol> 3. Cerrahi Prosedür <ol><li> Ayak veya kuyruk kısma anestezi derinliği izleyin. Orada bir yanıttır ve solunum yavaş ve daha fare uygun cerrahi düzlemdedir. </li><li> Sadece neredeyse bir No. 11 neşter bıçak kullanarak tek firma hareket kulaklar arasına gözlerin arkasında bir parasagital cilt kesi olun. Kenara cildi taşıyın ve hemostatla sağ tarafı klibi. </li><li> No: 11 neşter ve pamuk mat tarafı kullanılarak örten zarın Temizle kafatası aplikatör uçlu. Pamuklu çubuk% 0.9 tuzlu su çözeltisi içinde daldırma ile isteğe bağlı olarak, kafatası silin. Tamamen kurumasını bekleyin. </li><li> BiziKüçük bir cetvel ing, kalıcı bir kalem ile, sagital sütür (Şekil 1) yanal 1 mm koronal sütür ile kaudal ve 1 mm Kraniotomi sol kenarında Kraniotomi anterior sınırını çizecek. Sonra sagital ve koronal sütür, sırasıyla (Şekil 1) 4 mm kraniotomi hakkını ve kaudal sınırları işaretleyin. </li><li> 0.5 mm&#39;lik matkap ucu kullanarak, kalıcı marker anahat takip yavaş delerek kraniotomi yapmaya başlar. Tamamen kafatası yoluyla kırmak için değil emin olun. Basın hafifçe No. 5 forseps ile parietal kemiğin izole parçası üzerinde, zayıflık alanları basınca yol verecek. Inceltilmiş kemik çevre boyunca yeterince zayıf olduğunda, kemik parçası çıkarılmaya hazırdır. NOT: Araştırmacı deneyimleri zorluk tek parça kemik çıkarmadan, bu kemik yeterince sondaj esnasında inceltilmiş olmadığını göstermektedir. Facilita sonraki hayvanlarda daha kafatası delme dikkateKemik parçasının te kolay çıkarılması. </li><li> Bir 23 G iğne ile donatılmış 10 ml&#39;lik bir şırınga kullanarak, izole edilmiş kemik ve delinmiş alanı emmek için% 0.9 tuzlu su, küçük bir miktar uygulanır. </li><li> Stereotaksik ekipmana manipülatör kolunu takın. Rostrally bakan bıçak keskin tarafı ile prob tutucu için yeni bir No. 11 neşter bıçak takın. NOT: rostral ve kaudal dokular bıçağın keskin ve künt kenarları tecrübe olsa da, sırasıyla, delici yaralanma ile uyarılan mekanik hasar lezyonun ölçüde boyunca karşılaştırılabilir. Biz rostral ve kaudal bölümler arasında GFAP ifade veya çoğalması artışıyla da dahil olmak üzere reaktif gliozis en önemli özelliklerinden hiçbir kayda değer farklılıklar görüyoruz. </li><li> Yolumdan manipülatör kolunu tutarak, dikkatli 5/45 açılı forseps kullanılarak izole kemik kaldırın. Ful birinde geride kemik parçasını koparmak kaldıraç kullanarak, izole kemik ve asansörün yan içine forseps ucu takınl hareketi. NOT: Beyni bıçaklanma veya kafatası altında dura rahatsız etmemek için dikkatli olun. </li><li> Emdirilmiş jel köpük küçük bir parça alın ve kurumasını önlemek ve mevcut olabilecek herhangi bir kan emmek için çıplak beyin üzerine yerleştirin. </li><li> Jel köpük yerleştirildikten sonra, yerine manipülatör kolu salıncak ve jel köpük üzerinde Kraniotomi merkezine bıçağı ayarlayın. Jel köpük çıkarın ve uç dura dura delinmesiyle olmadan temas edene kadar bıçak indirin. Mark dorsal / stereotaksik dikey kolunda sürmeli ölçeği kullanılarak ventral koordinatlar. </li><li> Manipülatör kolunu kullanarak, yavaşça bıçağı beyin içine tam 3 mm indirin. Bu manipülatör kolunun üzerinde sürmeli ölçekli işaretleri kullanılarak elde edilir. Bıçak 5-10 saniye yerde kalmak için izin verir. Opposi geçmeden önce Kraniotomi rostral ve kaudal sınırları ulaşmasını sağlayan bıçağı üç kez kaudal bıçak eki rostral ile stereotaksik kol Taşıte sonu. NOT: Dura bıçağı takmadan önce kaldırılmaz. O | an sıçan dura, ~ aksine kalınlığı 7 80 um, fare dura önemli ölçüde daha incedir (sadece birkaç hücre tabakası kalınlığında) ve sokma esnasında bisturi için kayda değer bir direnç üretmez. Her hayvan tutarlı bir yaralanma almasını sağlamak için her bir fare için yeni bir neşter bıçak kullanın. </li><li> Yavaş yavaş beyin bıçağı çıkararak, stereotaksik kol yükseltmek. Bıçağın çıkarıldıktan sonra, hemen herhangi bir aşırı kan veya sıvının kadar emmek için beyin yüzeyindeki gelfoam başka bir parça koyun. </li><li> Bu arada, stereotaksik kol kaldırmak ve No. 11 neşter bıçak atmayın. Kanama durduktan sonra, gelfoam çıkarın. </li><li> Böyle etilon veya prolen olarak emilmeyen sütür, cildi dikilmesi yoluyla yarayı kapatın. Dikişler ameliyattan sonraki 9-10 gün çıkarılmalıdır. </li><li> Evde kafes fare dönün ve fare heatin yavaş yavaş kurtarmak için izinsıkıntı herhangi bir işaret için g ped ve monitör. Isofluorane kaynaklı anestezi Kurtarma tipik isofluorane çıkarıldıktan sonra 2-5 dakika içinde ortaya çıkar. Bu sternum recumbancy kazanmış kadar sahipsiz hayvan bırakmayın. </li><li> Hidrasyon sağlamak için laktat Ringer solüsyonu subkutan 0.5-1 ml yönetme. </li></ol> 4. Post-cerrahi Bakımı <ol><li> Yakından koloniye dönmeden önce anestezi kurtarma kadar ameliyat sonrası hayvanların izleyin. <ol><li> , Ameliyat sonrası ağrı ve rahatsızlığı azaltmak hemen prosedür takip ip enjeksiyon yoluyla 0.05-0.1 mg / kg buprenorfin yönetmek için. </li><li> Böyle kısıtlı hareketler, tımar eksikliği, ya da kilo kaybı gibi sıkıntı şiddetli belirtileri ameliyat sonrası 2-3 gün hayvanları gözlemleyin. Sıkıntı bu işaretlerin herhangi biri gösteren hayvanlar Euthanize ve çalışma çıkarın. </li></ol></li><li> Stan tarafından hayvanların euthanize, yaralı dokuların histopatolojisini incelemek içindard Intracardial perfüzyon. <ol><li> Kısaca, ketamin / ksilazin aşırı dozda hayvanlara anestezi, daha sonra intrakardiyak 15-20 ml% 0.9 NaCl emdirilmiştir veya karaciğer Deneyin sonunda% 4 paraformaldehit, 60 ml, ardından, kan temizlenene kadar. </li></ol></li><li> % 30 sukroz çözeltisi aktarmadan önce% 4 paraformaldehid içinde 2-4 saat boyunca beyin ve sonrası düzeltme teşrih. Garcia 8,9 açıklandığı gibi veya standart histolojik veya immünohistokimyasal prosedürlerle Bölüm 40-60 mikron bir kriyostat üzerinde beyinleri ve süreç. </li></ol>

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The authors have nothing to disclose.

Bu kafatası ya da altta yatan dura delme sırasında hasar görmemesi çok önemlidir. Patlak değil kafatası sağlamak için sondaj yaparken hafif baskı uygulayın. Dura sağlamak için kafatası parçasını kaldırma kemik ile kaldırdı değilken ek olarak, dikkatli olunmalıdır. ön beyin bıçak yaralanması modelleri burada MSS bir delici yaralanma nitelendirdi. Böyle FPI veya CCİ olarak TBI modellere göre daha az klinik çevrilebilir olsa da, ön beyin bıçak lezyon modeli ay...

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.

Bu prosedürü geçiren hayvanlar gerekmez, çünkü post-operatif bakım uzman, kısa veya uzun süreli zaman sağ kalım süreleri kolayca yaralanma sonrası akut veya kronik patolojiyi araştırmak için ihtiyaca göre, çalışmaya dahil edilmiştir. Böyle GFAP ve soma hipertrofisi artışı gibi reaktif gliozis temel özellikleri, gibi erken yaralanma sonrası 2-3 gün olarak görülebilir. Reaktif astrositlere için çoğalması zirve faz yaralanma 10 şu günlerde 3-5 sırasında. Aşağıda gösteril...

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.

Reaktif gliozis Tetikleme<em&gt; In Vivo</em&gt; Bir Önbeyin Stab Yaralanma tarafından

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 Görüntüleme.  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.

Bir In vivo</emDaralma indüklenen Sakatlık ve Kurtarma Non-invaziv İzleme> Kemirgen Model

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

Tıp

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.

Fare Shear stres İndüklenmiş Ateroskleroz Tetikleme Karotis Kol İmplantasyonu

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.

Hücre Takip için vivo 19 F MRG

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.

Rahim içi Telemetri Fare Kontraktil Basıncını Ölçmek<em&gt; İn 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.

Anastasis Vivo içinde CaspaseTracker Biyoalgılayıcı tarafından tespit

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.

İnsan Tümör ksenogratflarının gelen Fare Hücrelerinin tükenmesi Anlamlı Hedef Hücre Alt Analizi geliştirir

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.

Akut Akciğer Hasarı Açık Trakeostomi Gastrik Asit Aspirasyon Fare Modeli Maksimal Akut Ölümcül olmayan Akciğer Hasarı Sonuçlar

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.

Bir Fare Modelinde İnfrarenal Abdominal Aort Bağlama Ekipmanları tarafından tekrarlanabilir Arter denüdasyon Yaralanma

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.

Neuroregeneration periferik ve santral sinir sistemi çalışmak için bir Vivo Drosophila yaralanma modeli

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.

Doğrudan Intratracheal Lipopolysaccharide Instilasyon ile farelerde akut akciğer hasarının İndükleme

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