Os autores agradecer Dr. Lee Khai Pin, cabeça & Hospital consultor sênior, emergência infantil, KK feminina e infantil, 100 Bukit Timah Road, Singapore para o apoio administrativo com a produção de vídeo do artigo. Artigo reproduzido com a permissão de "Handheld Metal Detector para metálico estrangeiros corpo ingestão em pediátrica emergência"; Agosto de 2018:85 (8): 618-624; Gabriela Gabriele Hamzah, James vigília, Suraj Manickam Sashikumar Ganapathy: copyright The Indian Journal of Pediatrics.

O protocolo segue as diretrizes do Comitê de ética em pesquisa humana institucional. O Conselho de revisão institucional central desde a aprovação da ética para o estudo.
1. participante de triagem
<ol><li>Recrute pacientes que apresentam para o departamento de emergência pediátrico com história de ingestão de corpo estranho.</li>
	<li>Exclua os pacientes que estão com problemas respiratórios e implantaram dispositivos como ritmo fabricantes.</li>
</ol>2. preparar o paciente
<ol><li>Vesti o paciente em um roupão de hospital.</li>
	<li>Remova todos os itens que podem conter objectos metálicos, tais como roupas, óculos, brincos, pingentes e enfeites antes do exame para evitar interferência com o rastreio HHMD.</li>
	<li>Coloca o paciente no centro da sala longe de outras potenciais interferências metálicas.</li>
</ol>3. prepare o examinador e cuidadores
<ol><li>Retire todos os acessórios metálicos, tais como joias e relógio.</li>
	<li>Solicite o cuidador para remover todos os acessórios metálicos, tais como joias e relógio.</li>
</ol>4. metal Detector triagem
<ol><li>Use o HHMD (Figura 1) para realizar o rastreio.</li>
	<li>Confirme o status de trabalho da HHMD por suscitar um sinal positivo de audiovisual acenando sobre um pedaço de metal.</li>
	<li>Execute o HHMD digitalização em uma posição ereta para crianças maiores.</li>
	<li>Execute a varredura em uma posição ereta para infantes por ter a preensão do cuidador do paciente adequadamente HHMD.</li>
	<li>Segure as mãos do paciente e longe do corpo.</li>
	<li>Posição da cabeça do paciente em extensão para expor o pescoço.</li>
	<li>Digitalize a partir do nível do queixo ao nível da articulação do quadril para cobrir as áreas anatômicas do pescoço a pélvis.</li>
	<li>Fazer a varredura de uma forma de zig-zag em todo o corpo para garantir que cada área tem sido verificada.</li>
	<li>Digitalizar-ambos anteriormente e posteriormente como a espessura do paciente deve ser levada em consideração.</li>
	<li>Observe cuidadosamente o sinal de áudio-visual positivo durante a verificação da presença de MFBs.</li>
</ol>5. Realize a radiografia relevante
<ol><li>Ordem da radiografia relevante do pescoço, tórax ou abdômen, conforme a área detectada pela triagem de HHMD para os MFBs.</li>
	<li>Prosseguir com todas as exibições de raio-x (pescoço, peito e abdômen) se a MFB não foi detectado durante a triagem de HHMD e o índice clínico de suspeita de ingestão de corpo estranho é alta.</li>
</ol>6. gravar os resultados do teste
<ol><li>Recorde os resultados do teste no diário de bordo (Figura 2) criado especificamente baseiam no fluxo de trabalho (Figura 1).</li>
	<li>Correlacione o sinal positivo de HHMD audiovisual com o Marco ósseo subjacente no corpo, por palpar os seguintes Marcos ósseos proeminentes - entalhe esternal, xiphisternum, sínfise púbica e crista ilíaca bilateral.</li>
	<li>Grave o marco no contorno de referência anatômica do diário de bordo antes de prosseguir com o exame de raio-x.</li>
</ol>

<ol>
	<li>Lee, J., Ahmad, S., Gale, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+coins+ingested+by+children+using+a+handheld+metal+detector:+a+systematic+review.">Detection of coins ingested by children using a handheld metal detector: a systematic review.</a> Emergency Medicine Journal. 22 (12), 839-844 (2005).</li><li>Hesham A-Kader, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foreign+body+ingestion:+children+like+to+put+objects+in+their+mouth.">Foreign body ingestion: children like to put objects in their mouth.</a> World Journal of Pediatrics. 6 (4), 301-310 (2010).</li><li>Muensterer, O. 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A., Forte, V., Papsin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+commercially+available+metal+detector+for+the+localization+of+metallic+foreign+body+ingestion+in+children.">Use of a commercially available metal detector for the localization of metallic foreign body ingestion in children.</a> The Journal of Otolaryngology. 29 (4), 218-220 (2000).</li><li>Sacchetti, A., Carraccio, C., Lichenstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand-held+metal+detector+identification+of+ingested+foreign+bodies.">Hand-held metal detector identification of ingested foreign bodies.</a> Pediatric Emergency Care. 10 (4), 204-207 (1994).</li><li>Hamzah, H. B., James, V., Manickam, S., Ganapathy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handheld+Metal+Detector+for+Metallic+Foreign+Body+Ingestion+in+Pediatric+Emergency.">Handheld Metal Detector for Metallic Foreign Body Ingestion in Pediatric Emergency.</a> Indian Journal of Pediatrics. 85 (8), 618-624 (2018).</li><li>Doraiswamy, N. V., Baig, H., Hallam, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+detector+and+swallowed+metal+foreign+bodies+in+children.">Metal detector and swallowed metal foreign bodies in children.</a> Journal of Accident &amp; Emergency Medicine. 16 (2), 123-125 (1999).</li><li>Seikel, K., Primm, P. A., Elizondo, B. J., Remley, K. 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Os autores não têm nada para divulgar.

Metálico estrangeiros, ingestão de corpo em crianças é um problema comum e na maioria dos pacientes, isto é gerenciado conservadoramente7,16. Radiografias simples ou repetidas podem expor a população pediátrica da rádio sensível ao risco de radiação indesejada ao tentar localizar o corpo estranho metálico ingerido. Vários estudos têm descrito o uso de detectores de metais na ingestão de MFB em crianças, com sensibilidade a mais de 96% e especific...

Ingestão de corpo estranho é um problema generalizado entre as crianças, apresentando-se ao departamento de emergência pediátrico devido a natural natureza inquisitiva dos lactentes e crianças explorar objetos de degustação e engoli-los1,2. Estudos anteriores relataram que objetos metálicos constituíam 85% de todos os animais de corpo estranho na sua coorte de pacientes3,4. O objetivo do presente protocolo é avaliar a precisão e a viabilidade do uso de um detector de metais portátil para detectar corpos estranhos metálicos ingeridos em crianças. Propomos que, introduzindo o detector de metais portátil protocolo cedo no processo de triagem de crianças com alta suspeita de ingestão de corpo estranho metálico de triagem, o número de radiografias, sendo ordenado a localizar o corpo estranho metálico pode ser reduzido nesta população de radio-sensível.
Detector de metais portátil (HHMD) é um complemento barato, facilmente acessível que pode ajudar a agilizar a gestão de corpos estranhos metálicos ingeridos. Detector de metais é um dispositivo muito baixa frequência, que tem um receptor e um transmissor que detecta a alteração no seu campo eletromagnético causado pela presença de um objecto metálico nas suas proximidades3. Se um objeto metálico é dentro do campo, o campo eletromagnético é interrompido e o sensor irá disparar um alarme audiovisual sobre o dispositivo4. O exame de detector de metais pode ser executado completamente em menos de 2 minutos5. Portanto, a triagem de HHMD pode ser introduzida mais cedo no processo de triagem de crianças com alta suspeita de ingestão de corpo estranho metálico para localização topográfica.
As moedas são os mais comuns corpos estranhos metálicos engolido1,4,6,7. Devido à falta de bordas afiadas e a natureza geral de tóxicos, a maioria dos animais de moeda é gerenciada conservadoramente. Outros tipos de materiais comumente ingeridas incluem ímãs, pilhas de botão, brinquedos pequenos, amendoins, joias, botões, ossos, sementes e pipoca8,9. Impactação de corpos estranhos ocorre mais comumente no esôfago em três locais, nomeadamente do esfíncter esofágico superior, esôfago médio ao nível do arco aórtico e de esfíncter esofágico inferior10. Corpos estranhos metálicos (MFBs) localizados no esôfago podem ser assintomáticos em 40% dos pacientes11,12 e sua impactação prolongada pode levar a várias complicações, como perfuração esofágica, mediastinite, fístulas aorto-esofágico1,13,14,15, estenose traqueal e fístula traqueoesofágica. Outros locais de impactação de corpo estranho incluem piloro, duodeno e ileo-cecal junção10.
Realizar radiografias do pescoço, tórax e abdômen para diagnosticar a suspeita de ingestão de corpo estranho é uma prática generalizada nos departamentos de emergência5. Entre todos os tipos dos corpos estranhos ingeridos, apenas 10% são radiopacos10. Os exames radiológicos são demorados e têm um risco de exposição à radiação em crianças. Localização topográfica precisa ingeridos de objectos de metal é muito crucial para orientar a terapia. A introdução de HHMD triagem no processo de triagem pode ajudar a atenuar a estranhos pedidos de radiografias por topograficamente, localizando o corpo estranho metálico ingerido.
Este estudo avaliou a precisão de HHMD para localizar MFBs de forma sistemática e topográfica e determinar a eficácia e a viabilidade de HHMD na população pediátrica local. Também examinamos como bem esta radiação livre ferramenta de triagem foi aceita pelos pacientes e pais. Entre maio e julho de 2016, todos os pacientes consecutivos que apresentou ao departamento de emergência pediátrico com uma história de ingestão de corpo estranho foram incluídos no estudo. Foi obtido o consentimento do pai ou tutor legal. Pacientes com ingestão de corpo estranho alegada sistematicamente foram digitalizados usando HHMD. O padrão-ouro de cuidados para a identificação e localização de corpos estranhos metálicos ingeridos está realizando radiografias simples da área cervical, tórax e abdômen. Para facilitar a realização do estudo, um fluxo de trabalho padronizado foi criado (Figura 1). Um livro de registo especificamente criado com base no fluxo de trabalho foi usado para coletar os dados iniciais, que foram posteriormente transcritas para uma planilha (Figura 2). Os dados na seção de resultado são retratados como o número de casos, seguido por porcentagens.

<table>
	<tbody>
		<tr>
			<td>Hand held metal detector</td>
			<td>Garrett Super Scanner V</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>

handheld metal detector screening for metallic foreign body ingestion in children

Moedas são os corpos estranhos metálicos ingeridos mais comuns entre as crianças. O objetivo do presente protocolo é avaliar a precisão e a viabilidade do uso de um detector de metais portátil para detectar corpos estranhos metálicos ingeridos em crianças. Propomos que, através da introdução de rastreio de detector de metais portátil mais cedo no processo de triagem de crianças com alta suspeita de ingestão de corpo estranho metálico, o número de radiografias, sendo ordenado a localizar o corpo estranho metálico pode ser reduzido neste rádio sensível à população. O protocolo de estudo requer a seleção dos participantes para a história de ingestão de corpo estranho e exclusão de pacientes com insuficiência respiratória ou implantes metálicos. O paciente muda a roupa de hospital e itens que poderiam conter metal como óculos, brincos, pingentes e enfeites são removidos. O paciente é posicionado no centro da sala, longe de outras interferências metálicas. O status de trabalho do detector de metais portátil primeiro é confirmado por suscitar um sinal positivo de áudio-visual. Então o rastreio é feito em uma posição ereta com cabeça em extensão para expor o pescoço, do nível do queixo ao nível da articulação do quadril, para cobrir as áreas anatômicas do pescoço a pélvis em forma de zig-zag, tanto anteriormente como posteriormente. Um sinal positivo de audiovisual é cuidadosamente observado durante a verificação da presença de corpo estranho metálico. Radiografias pertinentes são ordenadas conforme a área detectada na triagem detector de metais. O detector de metais portátil foi capaz de identificar precisamente todas as moedas entre os ingerido corpos estranhos metálicos em nosso estudo. O detector de metais portátil consistentemente não poderia detectar não-moeda metálicos corpos estranhos. Este protocolo demonstra a precisão do detector de metais portátil na identificação e localização de moedas e moedas como corpos estranhos metálicos.

Os pesquisadores recrutaram 36 pacientes para este estudo observacional. Estes pacientes apresentaram-se ao departamento de emergência pediátrico com uma história de ingestão de corpo estranho. Entre estes, 28 pacientes apresentavam animais de corpo estranho metálico (Figura 3). Os restantes 8 pacientes ingeriu corpos estranhos não-metálicos, tais como ossos de peixes, satay stick e botão de plástico. As moedas eram o tipo mais comum de corpo estranh...

Watch this Scientific Journal Video about Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children at JoVE.com

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Aqui, apresentamos um protocolo para usar o detector de metais portátil, a tela a presença de ingerido corpos estranhos metálicos em crianças que apresentam ao departamento de medicina de emergência pediátrica, com uma história de ingestão de corpo estranho.

Triagem de Detector de metais portátil para ingestão de corpo estranho metálico em crianças

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

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10.6K visualizações.  KK women's and Children's Hospital. Aqui, apresentamos um protocolo para usar o detector de metais portátil, a tela a presença de ingerido corpos estranhos metálicos em crianças que apresentam ao departamento de medicina de emergência pediátrica, com uma história de ingestão de corpo estranho.

Vídeo: Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

Screening alto teor em Doenças Neurodegenerativas

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

Medicina

Eye Tracking Young Children with Autism

The rise of accessible commercial eye-tracking systems has fueled a rapid increase in their use in psychological and psychiatric research. By providing a direct, detailed and objective measure of gaze behavior, eye-tracking has become a valuable tool for examining abnormal perceptual strategies in clinical populations and has been used to identify disorder-specific characteristics1, promote early identification2, and inform treatment3. In particular, investigators of autism spectrum disorders (ASD) have benefited from integrating eye-tracking into their research paradigms4-7. Eye-tracking has largely been used in these studies to reveal mechanisms underlying impaired task performance8 and abnormal brain functioning9, particularly during the processing of social information1,10-11. While older children and adults with ASD comprise the preponderance of research in this area, eye-tracking may be especially useful for studying young children with the disorder as it offers a non-invasive tool for assessing and quantifying early-emerging developmental abnormalities2,12-13. Implementing eye-tracking with young children with ASD, however, is associated with a number of unique challenges, including issues with compliant behavior resulting from specific task demands and disorder-related psychosocial considerations. In this protocol, we detail methodological considerations for optimizing research design, data acquisition and psychometric analysis while eye-tracking young children with ASD. The provided recommendations are also designed to be more broadly applicable for eye-tracking children with other developmental disabilities. By offering guidelines for best practices in these areas based upon lessons derived from our own work, we hope to help other investigators make sound research design and analysis choices while avoiding common pitfalls that can compromise data acquisition while eye-tracking young children with ASD or other developmental difficulties.

Eye tracking has long been used to study gaze patterns in typically-developing individuals, but recent technological advancements have made its use with clinical populations, including autism, more feasible. While eye-tracking young children with autism can offer insight into early symptom manifestations, it involves methodological challenges. Suggestions for best practices are provided.

Rastreamento ocular crianças com autismo

The rise of accessible commercial eye-tracking systems has fueled a rapid increase in their use in psychological and psychiatric research. By providing a ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Protocolo para Longa Duração Hipertermia corpo inteiro em Ratos

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory rate, salivation, perspiration and renal function. The ANS has two main branches: the sympathetic nervous system, preparing the human body for action in times of danger and stress, and the parasympathetic nervous system, which regulates the resting state of the body.
ANS activity can be measured invasively, for instance by radiotracer techniques or microelectrode recording from superficial nerves, or it can be measured non-invasively by using changes in an organ's response as a proxy for changes in ANS activity, for instance of the sweat glands or the heart. Invasive measurements have the highest validity but are very poorly feasible in large scale samples where non-invasive measures are the preferred approach. Autonomic effects on the heart can be reliably quantified by the recording of the electrocardiogram (ECG) in combination with the impedance cardiogram (ICG), which reflects the changes in thorax impedance in response to respiration and the ejection of blood from the ventricle into the aorta. From the respiration and ECG signals, respiratory sinus arrhythmia can be extracted as a measure of cardiac parasympathetic control. From the ECG and the left ventricular ejection signals, the preejection period can be extracted as a measure of cardiac sympathetic control. ECG and ICG recording is mostly done in laboratory settings. However, having the subjects report to a laboratory greatly reduces ecological validity, is not always doable in large scale epidemiological studies, and can be intimidating for young children. An ambulatory device for ECG and ICG simultaneously resolves these three problems.
Here, we present a study design for a minimally invasive and rapid assessment of cardiac autonomic control in children, using a validated ambulatory device 1-5, the VU University Ambulatory Monitoring System (VU-AMS, Amsterdam, the Netherlands,<a href="http://www.vu-ams.nl" target="_blank"> www.vu-ams.nl</a>).

Measuring Cardiac Autonomic Nervous System ANS Activity in Children

Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.

Medindo a atividade cardíaca Nervoso Autônomo (SNA) Sistema em Crianças

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Triagem para Modificadores Melanoma usando um modelo de tumor Zebrafish autóctone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel de alta resolução<em&gt; In vivo</em&gt; Técnica de Imagem para estudar a resposta dinâmica de estruturas intracranianas ao crescimento do tumor e Terapêutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel<em&gt; In vivo</em&gt; Técnica Gene Transferência e<em&gt; In vitro</em&gt; Ensaios baseados em células para o Estudo da perda óssea em Distúrbios Osteomusculares

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Normotérmica<em&gt; Ex Vivo</em&gt; Perfusão rim para a Preservação do rim Os enxertos antes do transplante

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Heterotópico Renal autotransplante em um modelo suíno: Um Protocolo Passo-a-Passo

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

-Nervo poupadores Mid-obstrução uretral (Nemo) em fêmeas pequenos roedores

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...