We thank Dr. John Ohlfest and Dr. Stacey Decker13 for the generous gift of plasmids and for the training provided to master this method. We thank Marta Dzaman for help with standardizing the Hematoxylin-Eosin staining procedure of paraffin-embedded sections. We also thank Molly Dahlgren for perusing this manuscript and providing helpful suggestions. This work is supported by NIH/NINDS grants to MGC and PRL, Leah&#8217;s Happy Hearts Foundation grant to MCG and PRL. Alex&#8217;s Lemonade Foundation young Investigator Award and the St. Baldrick&#8217;s Foundation Fellowship to CK.

<ol>
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The authors have nothing to disclose.

Neste artigo, que detalhe um método versátil e reprodutível para a geração de novos modelos de GBM utilizando integração mediada SB transposase- de ADN plasmídeo oncogénica em células vizinhas da zona subventricular dos ratinhos neonatais. Nós apresentamos um protocolo para gerar plasmídeos transposon com novos genes de interesse, ilustrar como monitorar a progressão dos tumores em animais vivos, e como caracterizar características histopatológicas e imuno-histoquímica destes tumores. <p class="jove_...

O glioblastoma multiforme (GBM) é o (60%) de tumor cerebral primário mais comum em adultos, com uma sobrevivência média de 15-21 meses, quando tratados com cirurgia, radioterapia, quimioterapia e 1. Os novos tratamentos para GBM são imperativos. Terapias experimentais requerem testes em modelos animais que se reproduzem de forma adequada as principais características da doença humana. Estratégias para induzir GBM em roedores incluem mutagênese química com agentes alquilantes, germline ou alterações genéticas somáticas, ou transplante utilizando linhas celulares de glioma 2. Os modelos mais correntemente utilizados empregam a implantação de linhas de células de glioma, ou ortotopicamente, para dentro do cérebro ou por via subcutânea, utilizando células singeneicas em animais com genótipo idêntico; ou xenogénicas, linhas mais comumente humanos de células de GBM, implantados em camundongos 3 imunológico comprometido. Os xenoenxertos oferecem muitas vantagens para o estudo de tumores intracranianos: conveniência de reprodutibilidade, Standataxas de crescimento rdized, hora da morte e localização do tumor. No entanto, estes modelos têm limitações devido à abordagem cirúrgica artificial, invasivo utilizado para implantes e limitada capacidade de reproduzir com precisão histológico apresenta característica de GBM humano (OMS grau IV): necrose pseudo-pallisading, atipias nucleares, invasão difusa, a proliferação de micro-vascular e a formação de anormalidades vasculares glomerulóides 4-7. Indução de GBM por alterar o genoma de células somáticas com ADN oncogénico, ou com vectores virais 8-12, ou com integração mediada por transposão 13, reproduz mais de perto a etiologia da doença humana e recapitula características histopatológicas de GBM humano. Historicamente, os tumores do SNC foram classificados com base na célula percebida de origem, o que foi observado, seria um fator preditivo de sobrevivência 14. GBMs são classificadas em primárias e secundárias. Emergentes evidência today aponta para a natureza altamente heterogêneo de glioblastoma 15 primário. GBM secundário (15%), o resultado da transformação maligna de astrocitoma de baixo grau (OMS grau I) e astrocytomas anaplasic (OMS grau II), estão associados com início mais precoce da doença, melhor prognóstico e um padrão de &quot;proneural&quot; da expressão gênica , enquanto que GBM primário (85%) mostram um início tardio, o prognóstico pobre e glial (clássica), neural ou padrões de expressão mesenquimais. Se estes padrões de expressão de genes correlacionam-se com a célula de origem real do tumor é ainda a ser activamente investigados. A acumulação de dados mostra que a combinação de mutações genéticas associadas com GBMs são preditivos para a sobrevivência. Por exemplo, a perda de heterozigose (LOH) de cromossomos 1p / 19q, mutações IDH1, PDGFRα ampliações, estão associados com GBMs secundárias, padrão de expressão proneural e melhor prognóstico, enquanto que a superexpressão de EGFR, Notch e Sonic hedgehog ativação da via, Nf1 e PTEN perda e mutações do p53 estão correlacionados com neural, clássica, ou GBM primário mesenquimais e pior prognóstico 16,17. O advento dos projetos de seqüenciamento em larga escala e da acumulação de numerosos espécimes de pacientes disponíveis para teste traz uma riqueza de novas informações no que diz respeito a mutações genéticas e os caminhos envolvidos na GBM patogênese e progressão ea possibilidade de medicina individualizada, onde terapias pode ser adaptado especificamente para as anormalidades genéticas do paciente. Em última análise, para avaliar o valor preditivo destas mutações e caminhos de uma forma sistemática, e para testar possíveis tratamentos para cada caso, requer modelos animais de GBM com alterações genéticas pré-determinados. Transposon integração mediada de DNA genômico oferece uma abordagem viável. O sistema transposon Bela Adormecida, membro da classe / mariner Tc1 de transposons, foi &quot;acordado&quot; (construído) em um proce multi-passoss de mutagênese sítio específico de um gene transposase salmonóide, que se tornou mais dormente de 10 milhões de anos atrás 18,19. Em essência, os transposons de ADN flanqueadas por sequências específicas (repetições invertidas / repetições directas: IR / DR) podem ser integrados no genoma de uma maneira &quot;corta e cola&quot; por meio da actividade da transposase sono de beleza. A transposase reconhece os extremos dos sites de RI, extirpa o transposão e integra aleatoriamente em um outro local de ADN entre as bases de T e A, bases que são duplicados em cada uma das extremidades do transposão durante transposição (Figura 1a). O sono transposase beleza é composta por três domínios, um domínio de ligação de transposões, uma sequência de localização nuclear e um domínio catalítico. Quatro moléculas de transposase são necessárias para aproximar as duas extremidades do transposão em conjunto e permitir a transposição, no entanto, se demasiadas moléculas de transposase estiver presente, pode dimerizar e tetramerize para inibira reacção de transposição 20. Uma reação transposição eficiente requer uma ótima relação de transposase de transposons. O ADN que codifica para a transposase pode ser entregue no mesmo plasmídeo com o transposão (em cis) ou num plasmídeo diferente (em trans). Para assegurar a relação óptima entre a transposase e os transposões, um promotor com actividade adequada pode ser escolhido para a expressão da transposase (para o modelo de &quot;cis&quot;) ou a relação de plasmídeos na solução injectável podem ser optimizados (para o modelo de &quot;trans&quot;). O sistema transposon Bela Adormecida pode ser utilizado com sucesso para a genómica funcional, mutagênese insercional, transgenia e terapia genética somática 21. Sendo uma construção sintética re-projetado para uma molécula funcional a partir de uma variante salmonóide dormente, Sleeping Beauty transposase não se liga a outros transposons em seres humanos ou outros mamíferos 20. Desde a sua descoberta, a engenharia molecular tem enhanced transposição a eficácia do sistema de transposão SB através da modificação das sequências IR e adição de dinucleótidos TATA flanqueando o transposão, resultando nos transposões PT2. Estes transposons tem otimizado ligação do SB ao site IR e aumento da eficácia da excisão. A transposase SB também passou por melhorias significativas; transposase utilizado em experiências aqui apresentadas é o SB100X, uma transposase hiperactivo gerado por uma estratégia de arranjo combinatório de ADN seguido por um rastreio genético em larga escala em células de mamífero 22. Neste relatório apresentamos um método rápido, versátil e reprodutível para induzir intrínseca GBM em camundongos com, integração mediada por transposon não-viral de DNA plasmídeo em células da zona sub-ventricular de ratos neonatal 23. Nós apresentamos uma maneira simples de criar transposons com novos genes passíveis de integração genômica usando o sono transposase Beleza e demonstrar como monitorar a captação plasmídeo eprogressão da doença usando a bioluminescência. Nós também caracterizar as características histológicas e imuno-histoquímica do GBM reproduzido com este modelo. Além disso, apresentamos um método rápido para gerar linhas de células de GBM primárias destes tumores. O modelo de beleza do sono, em que os tumores são induzidos a partir de células originais ao animal, permite a avaliação funcional do papel dos genes candidatos GBM na indução e a progressão de tumores. Este sistema também é bem adequado para o teste de tratamentos novos GBM, incluindo terapias imunológicas em ratos imuno-competentes, sem a necessidade de procedimentos cirúrgicos invasivos inflamatórias, que podem alterar o microambiente local.

<table border="0" cellpadding="0" cellspacing="0" width="952">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:199px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:367px;">Stoelting&#39;s Lab Standard with Rat and Mouse Adaptors</td>
			<td style="width:199px;">Stoelting</td>
			<td style="width:119px;">51670</td>
			<td style="width:267px;">Other companies produce similar frames, any of them with small mouse adaptors are suitable&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:367px;">Quintessential Stereotaxic Injector (QSI)</td>
			<td style="width:199px;">Stoelting</td>
			<td style="width:119px;">53311</td>
			<td style="width:267px;">Injections can be made without it, ,but&#160;&#160;&#160; the automatic injector allows for increased reproducibility and convenience</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:367px;">10 &#956;L 700 series hand fitted MICROLITER syringe</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:119px;">Model 701</td>
			<td style="width:267px;">This syringe model will deliver from 0.5 to 500 ul of solution reliably. Other syrnges (fro example 5 ul, Model 75) may also be used</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:367px;">30 gauge gauge hypodermic needle 
			(1.25&#34;, 15o bevel)&#160;</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:119px;">S/O# 197462</td>
			<td style="width:267px;">This needle is ideal to pierce the skin and the skull of a neonatal mouse without the need of other invasive procedures. If it gets dull (doesn&#39;t easily enter the skin), it needs to be replaced.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">in vivo-jetPEI</td>
			<td style="width:199px;">Polyplus Transfection</td>
			<td style="width:119px;">201-10G</td>
			<td style="width:267px;">Aliquot in small volumes and keep at -20oC&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">D-Luciferin, Potassium Salt</td>
			<td style="width:199px;">Goldbio.com</td>
			<td style="width:119px;">LuckK-1g</td>
			<td style="width:267px;">Other sources of firefly lucifierase are just as adequate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Hematoxylin Solution, Harris Modified</td>
			<td style="width:199px;">Sigma Aldrich</td>
			<td style="width:119px;">HHS128-4L</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:367px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:199px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">62550-01</td>
			<td style="width:267px;">for embedding brains for cryosectioning and immunohistovhemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Advanced DMEM/F-12, no glutamine</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">12634-010</td>
			<td style="width:267px;">for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">B-27&#168; Supplement (50X), serum free&#160;</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:267px;">serum free supplement for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">N2 Supplement (100x)</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:267px;">serum free supplement for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Normocyn</td>
			<td style="width:199px;">InvivoGen</td>
			<td style="width:119px;">ant-nr-1</td>
			<td style="width:267px;">anti-mycoplasma agent for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Recombinant Human EGF</td>
			<td style="width:199px;">PEPROTECH</td>
			<td style="width:119px;">AF-100-15</td>
			<td style="width:267px;">growth factor supplement for the culture of&#160; neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Recombinant Human FGF-basic</td>
			<td style="width:199px;">PEPROTECH</td>
			<td>100-18B</td>
			<td style="width:267px;">growth factor supplement for the culture of&#160; neurospheres</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">HyClone HyQTase&#160; Cell Detachment Reagent&#160;</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:119px;">SV3003001</td>
			<td style="width:267px;">for the dissociation of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kimble-Chase Kontes Pellet Pestle</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>K749510-0590</td>
			<td style="width:267px;">for the dissociation of freshly dissected tumors</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Falcon Cell Strainers mesh size 70um</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>08-771-2</td>
			<td style="width:267px;">for generating single cell suspensions of dissociated neurospheres</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Xylenes Histological grade</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">C8H10</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Protocol Harris Hematoxylin Mercury free ( acidified)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">245-678</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Protocol Eosyn Y Solution ( intensified)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">314-631</td>
			<td style="width:267px;"></td>
		</tr>
	</tbody>
</table>

transposon mediated integration of plasmid dna into the subventricular zone of neonatal mice to generate novel models of glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Para caracterizar as alterações histopatológicas de glioblastomas induzidas-SB, ratinhos neonatais C57 / BL6 foram injectados em P1 com um plasmídeo que codifica a luciferase (pT2 / SB100x-Luc), em combinação com plasmídeos codificando os transposons com ADN oncogénico, ou seja, NRAS (pT / CAGGS -NRASV12) e SV40 LGT (pT / CMVSV40-LGT) (Figura 3c) ou um plasmídeo que codifica uma p53 curto hairpin PDGFβ e com um repórter GFP (pT2shp53 / GFP4 / mPDGFβ) em combinação com NRAS <strong...

Watch this Scientific Journal Video about Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma at JoVE.com

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

Transposon Mediated Integração de DNA plasmídeo na zona subventricular de Neonatal Ratos para gerar modelos novela de Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

transposon-mediated-integration-plasmid-dna-into-subventricular-zone

Research

JoVE Journal

Medicine

12.8K visualizações.  University of Michigan School of Medicine. Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

Vídeo: Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Uso de Modelo Animal de Sepse para avaliar novas terapias à base de plantas

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Medicina

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

Identificação de<em&gt; A Bela Adormecida</em&gt; Inserções de transposões em tumores sólidos utilizando Linker mediada por PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

Imagem-guiado Entrega reforçada-Convecção em Modelos gel de agarose do Cérebro

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Injeção Orthotopic de células cancerosas da mama na mamária Fat Pad de Ratos para o Estudo de crescimento tumoral.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rato Transplante Renal: Modelos de rejeição de enxertos

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Um Modelo de Leitão Neonatal encefalopatia hipóxico-isquêmica

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Modelos cirúrgicos de refluxo gastroesofágico com Ratos

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Infarto do miocárdio em ratos neonatais, um modelo de regeneração cardíaca

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

Na Vivo Photolabeling de células no cólon para avaliar o potencial migratória das células hematopoiéticas em camundongos Neonatais

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Full-Endoscopic Isolation Zone Technique for the Treatment of Lumbar Disc Herniation

Lumbar disc herniation (LDH) is a type of serious sinus or sciatic nerve dysfunction caused by nucleus pulposus protrusion and annulus fibrosus tears. Its clinical symptoms often include severe low back pain, limited lumbar movement, sciatic nerve pain in the lower limbs, and even cauda equina syndrome. The common treatment for LDH is a conservative treatment scheme involving medicine, rest, and physical therapy. However, if the conservative treatment scheme is ineffective, a surgical treatment approach is adopted. Traditional open lumbar surgery has some disadvantages, including the potential for severe surgical trauma, severe blood loss during the operation, instability of the lumbar spine, and loss of the lumbar motor unit. Among the minimally invasive surgical schemes, full-endoscopic spine surgery (FESS) is undoubtedly the most appropriate and has the advantages of minimal trauma, high safety, quick postoperative recovery, and the retention of the stable structure and the motor unit of the lumbar spine. However, simultaneously, incomplete removal of the nucleus pulposus and residual nerve dysfunction after surgery can occur. To avoid these shortcomings, we studied a specific spinal endoscopy technique, the "isolation zone" surgical strategy, which can effectively block the pain from the nerve conduction pathway by completely relieving the nerve compression and nerve dysfunction through the orderly treatment of the protruding nucleus pulposus, the fissure of the annulus fibrosus, the sinus nerve, and the surrounding inflammatory soft tissues.

Here, we present a protocol for the &#34;isolation zone&#34; technique in the treatment of lumbar disc herniation (LDH) under full-endoscopic spine surgery (FESS), including intervertebral foramen formation, targeted catheterization, nucleus pulposus resection, and annulus fibrosus formation, which together completely block the pain from the nerve conduction pathway.

Técnica de zona de isolamento endoscópica total para o tratamento da hérnia discal lombar

Lumbar disc herniation (LDH) is a type of serious sinus or sciatic nerve dysfunction caused by nucleus pulposus protrusion and annulus fibrosus tears. Its ...