Teste de calor Dor usando o Thermostimulator TSA II 1. Introdução <ol><li> Este procedimento usa o TSA II, um disponível comercialmente analisador termo-sensorial que proporciona estímulos de calor para a pele com uma thermode (Medoc Advanced Medical Systems, Durham, NC). A unidade central gera e transmite o sinal para o thermode. Este sinal é controlado por software proprietário normalmente armazenados em um laptop conectado à unidade central. Também ligado à unidade central é um controle de mão que permite que um objeto de estudo para parar o aquecimento do thermode. </li><li> Sete estímulos de calor seqüencial e idêntica são entregues, com intervalos inter-estímulo de 30 anos. A temperatura basal thermode é fixado em um confortável 35 º C. A taxa na qual o thermode aquece é fixada em 1 º C / s, enquanto a taxa a que esfria é fixada em 10 º C / s. A temperatura thermode máxima é de 52 º C. </li></ol> 2. Preparação e treinamento os sujeitos <ol><li> Para preparar indivíduos para o banco de ensaio de calor a dor-los confortavelmente em uma poltrona reclinável que fornece suporte adequado para a cabeça, braços e pernas. A cadeira também deve permitir fácil acesso aos locais do corpo examinado. </li><li> A instalação de teste deve ser a uma temperatura ambiente confortável, e proporcionar um ambiente bastante neutro e sem nenhuma distração. </li><li> Antes de iniciar o experimento, todos os participantes devem ser bem treinados em todos os aspectos do teste de dor de calor. O treinamento pode ocorrer a qualquer momento antes do experimento. </li><li> Interações entre os sujeitos do estudo e os investigadores devem ser limitados àqueles necessários para a coleta de dados. Brincando e conversa são desencorajados porque eles podem comprometer a consistência dos dados. Sujeitos do estudo são informados de que interações intencionalmente ser um pouco monótona e neutros durante os experimentos. </li><li> Manter a instruções claras, simples e consistente. Enfatizar que a dor é subjetiva, e não há respostas certas ou erradas. É mais importante ser consistente. Definir o limiar de dor de calor como o ponto entre &quot;estar prestes a ser dolorosa&quot; e &quot;só se tornou doloroso.&quot; </li><li> É fundamental que um sujeito sabe exatamente o que esperar durante uma sessão de testes. Qualquer &quot;surpresa&quot; pode resultar em menos dados consistentes, porque confundidores tais como a ansiedade pode ser gerada. Por exemplo, se sete estímulos de dor de calor são usados ​​para determinar o limiar de dor de calor, o assunto deve saber isso. </li><li> A qualquer momento, o assunto pode parar a qualquer thermode de ficar mais quente, pressionando o botão de um controle de mão. Estar no controle irá reduzir a ansiedade de um sujeito e tornar os dados mais confiáveis. </li><li> A sessão de treinamento oferece uma oportunidade para avaliar o desempenho de um assunto estudo. Pode ser o melhor para treinar indivíduos durante 2-4 ciclos de ensaio para que seus limites de dor individuais variam de acordo com não mais de 0,8 ° C, e, idealmente, por não mais que 0,6 ° C entre os dois últimos ciclos de ensaio. Indivíduos que não conseguem executar de forma consistente são normalmente excluídos do estudo. </li></ol> 3. Determinar o limiar de dor <ol><li> Para começar a dor ciclos de testes de limiar marca o posicionamento do thermode na pele do sujeito. Neste experimento, a mesma lesão de pele inflamada usado para microdiálise serão testados para a dor de aprender sobre bioquímicos liberado em resposta ao teste de dor. </li><li> Então, iniciar cada ciclo, colocando o thermode para a área de pele marcada. Coincidir com a posição do thermode com as marcas o mais próximo possível. Permitir que a temperatura entre a pele eo thermode para equilibrar a 35 ° C - uma temperatura que seguramente causará nenhuma dor. Isso levará cerca de 5 segundos e será indicado por uma leitura de 35 ° C na tela do computador. </li><li> Antes de começar, dar o controle de mão para o assunto. </li><li> Use a pressão constante e suave para manter toda a superfície do thermode em pleno contacto com a pele do sujeito durante o teste. </li><li> Agora iniciar o algoritmo de teste de pré-programadas. O thermode vai começar a aquecer em 1 ° C / s. Assim que o sujeito experimenta a dor, eles vão pressionar o dispositivo de mão. </li><li> Após a pressionar o botão no dispositivo de mão, a dor de temperatura causando thermode será automaticamente registrado e vai começar a diminuir a uma taxa de 10 ° C / s para baixo a sua temperatura basal de 35 ° C. </li><li> A sonda de temperatura será automaticamente mantida a 35 ° C por 30 s antes de começar a subir novamente em 1 ° C / s, até que um assunto de novo aperta o botão do dispositivo de mão. Pouco antes de a temperatura thermode está prestes a aumentar novamente, lembrar o assunto que isso está prestes a acontecer. Isto irá manter o assunto focado. </li><li> Esta seqüência será repetida até sete gravações de temperatura foram registradas. Calcular a média destes tempeleituras ratura, este é o limiar de dor de calor. </li></ol> Teste Dor mecânica com sondas de pressão pontuado 1. Introdução <ol><li> Usamos feitos calibrado sondas de pressão pontuado, porque eles fornecem resultados mais consistentes do que os tradicionalmente usados ​​e disponíveis comercialmente cabelos von Frey. Sondas são feitos de idêntica cilíndrica fios de aço inoxidável (240 diâmetro mm) montados em barras de cobre de diferentes pesos moleculares (1, 2, 4, 8, 16, 32, 49, 64 e 81 gramas) que se movem livremente dentro de uma ampla mão- realizada tubo de alumínio. </li><li> O mesmo condições gerais experimental descrito para o teste de calor a dor se aplicam a testes de dor mecânica. Os participantes devem estar bem preparados e treinados. É especialmente importante para tranquilizar assuntos que as sondas estão sem corte e não perfurar a pele. Isso vai ajudar os sujeitos a relaxar e fornecer dados consistentes. </li></ol> 2. Determinar o limiar de dor mecânica <ol><li> Para determinar o limiar de dor mecânica, primeiro aplique a ponta do fio de aço da mais leve sonda perpendicularmente à pele no centro da lesão experimentalmente inflamado. </li><li> O tubo de alumínio de mão é então abaixada para a pele sem tocá-lo. Desta forma, todo o peso da haste de cobre no interior do tubo de alumínio repousa sobre a ponta do fio de aço. </li><li> O objeto de estudo, em seguida, indica se a sonda provoca dor, dizendo &quot;sim&quot; ou &quot;não&quot;. </li><li> Se as respostas assunto &quot;não&quot;, em seguida, a mesma sonda ou sonda mais pesado que vem será utilizado seguinte. Este algoritmo é chamado de uma seqüência de estimulação ascendente. Aleatoriamente determinar se a sonda ao lado é mais pesado ou com o mesmo peso. Isto irá manter um sujeito cego para o algoritmo. </li><li> Aplicar as sondas em seqüência ascendente até que um sujeito responde &quot;sim&quot;. O peso médio dos últimos dois detecções desta série é gravada como o ponto de deflexão em primeiro lugar. </li><li> Após as respostas assunto &quot;sim&quot;, começar a usar uma seqüência de estimulação descendente, e usar um isqueiro ou sonda a sonda mesmo de antes. Aleatoriamente determinar se a sonda próxima é mais leve ou do mesmo peso. Isto irá manter um sujeito cego para o algoritmo. </li><li> Aplicar as sondas em uma seqüência descendente, até as respostas de assunto &quot;não&quot;. O peso médio dos últimos dois sondas nesta seqüência constituem o ponto de deflexão segundo. </li><li> Quando o assunto respostas &quot;não&quot;, voltar para uma seqüência de estimulação ascendente. Alternam entre seqüências ascendentes e descendentes até sete pontos de deflexão são gravadas. A média desses sete pontos é o limiar de dor mecânica. </li></ol>

<ol>
	<li>Angst, M. S., Clark, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comment+on+Koltzenburg+et+al.:+Differential+sensitivity+of+three+experimental+pain+models+in+detecting+the+analgesic+effects+of+transdermal+fentanyl+and+buprenorphine.">Comment on Koltzenburg et al.: Differential sensitivity of three experimental pain models in detecting the analgesic effects of transdermal fentanyl and buprenorphine.</a> Pain. 126, 165-174 (2006).</li><li>Angst, M. S., Clark, J. D., Carvalho, B., Tingle, M., Schmelz, M., Yeomans, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+in+human+skin+in+response+to+experimental+inflammation,+noxious+stimulation,+and+administration+of+a+COX-inhibitor:+A+microdialysis+study.">Cytokine profile in human skin in response to experimental inflammation, noxious stimulation, and administration of a COX-inhibitor: A microdialysis study.</a> Pain. , (2008).</li><li>Angst, M. S., Koppert, W., Pahl, I., Clark, J. D., Schmelz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+infusion+of+the+mu-opioid+agonist+remifentanil+in+humans+causes+hyperalgesia+during+withdrawal.">Short-term infusion of the mu-opioid agonist remifentanil in humans causes hyperalgesia during withdrawal.</a> Pain. 106, 49-57 (2003).</li><li>Lotsch, J., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mu-opioid+agonist+remifentanil+attenuates+hyperalgesia+evoked+by+blunt+and+punctuated+stimuli+with+different+potency:+a+pharmacological+evaluation+of+the+freeze+lesion+in+humans.">The mu-opioid agonist remifentanil attenuates hyperalgesia evoked by blunt and punctuated stimuli with different potency: a pharmacological evaluation of the freeze lesion in humans.</a> Pain. 102, 151-161 (2003).</li></ol>

Este vídeo demonstra algoritmos para avaliar o calor eo limiar de dor mecânica em pele inflamada de voluntários humanos. A percepção da dor é subjetiva. Recolha de dados adequado da dor requer uma instalação de testes adequados (por exemplo, tranquilo e confortável), a formação completa dos participantes do estudo com dados in-built medidas de controle de qualidade (teste-reteste), seguir rigorosamente um protocolo de estudo padronizado e evitar fatores de confusão (por exemplo, ansiedade, estresse). Confiável de dados subjetivos sobre a sensibilidade à dor, emparelhado com intervenções como a administração de um medicamento analgésico e da recolha de dados mecanicista, como a liberação local de mediadores nociceptivos fornecer informações valiosas sobre os mecanismos da dor, e os meios para prevenir ou atenuar-lo.

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<td>TSA-II Neuro Sensory Analyzer</td>
<td>Tool</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Puntuated Pressure Probes</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
<td>Alternatively, commercially available von Frey Hairs can be used.</td>
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determining heat and mechanical pain threshold in inflamed skin of human subjects

Em um artigo anterior no Journal of Experiments visualizada demonstramos técnicas de microdiálise pele para a recolha de tecidos específicos bioquímicos nociceptiva e inflamatória em humanos. Neste artigo vamos mostrar a dor de teste de paradigmas que muitas vezes são usados ​​em conjunto com os procedimentos de microdiálise. Combinando testes dor com microdiálise fornece o elo crítico entre os dados comportamentais e bioquímicos que permite identificar bioquímicos principais responsáveis ​​pela geração e propagação da dor. Dois modelos de dor evocando em pele inflamada dos participantes do estudo humano são mostrados. O primeiro modelo evoca a dor com a ajuda de estímulos de calor. Dor de calor evocado como descrito aqui é predominantemente mediado pelas pequenas organizações não-mielinizadas periférico as fibras nervosas nociceptivas (fibras C). O segundo modelo evoca a dor através de estímulos pressão pontuado. Dor de pressão pontuado evocado é predominantemente mediado por pequenas mielinizadas periférico as fibras nervosas nociceptivas (fibras A-delta). Os dois modelos são mecanicamente distintas e de forma independente examine processamento nociceptivo pelas duas grandes populações de fibras nervosas periféricas envolvidas na sinalização da dor. Dor de calor é evocada com a ajuda da TSA II, um disponível comercialmente analisador termo-sensoriais (Medoc Advanced Medical Systems, Durham, NC). Configuração de estímulo e de entrega é feita com auxílio de software específico. Térmodos variam em tamanho e forma, mas em princípio, por uma placa de metal que podem ser aquecidas ou resfriadas a taxas diferentes e por diferentes períodos de tempo. Algoritmos de avaliação de dor provocada pelo calor são múltiplas. Nos experimentos mostrados aqui, os participantes do estudo são solicitados a indicar em que ponto eles começam a sentir dor enquanto o thermode em contato com a pele é aquecida a uma taxa predeterminada a partir de uma temperatura que não evoca a dor. A temperatura thermode em que um sujeito começa a experimentar a dor constitui o limiar de dor de calor. Dor mecânica é evocado com sondas pontuado. Tais sondas estão disponíveis comercialmente a partir de vários fabricantes (cabelos von Frey). No entanto, a precisão de von Frey cabelos tem sido criticada e muitos investigadores uso custom made sondas de pressão pontuado. Nos experimentos mostrados aqui oito feitos por sondas pontuado de pesos diferentes são aplicadas em ordem consecutiva, um procedimento chamado up-down algoritmo, para identificar pontos de deflexão perceptional, ou seja, uma mudança de sentindo nenhuma dor de sentir dor ou vice-versa. O peso médio causando uma deflexão perceptional constitui o limiar de dor mecânica.

Watch this Scientific Journal Video about Determining heat and mechanical pain threshold in inflamed skin of human subjects at JoVE.com

Determining heat and mechanical pain threshold in inflamed skin of human subjects

Algoritmos de avaliar o limiar da dor de calor e mecânica em pele inflamada experimentalmente humana dos sujeitos do estudo são mostradas. Os dois paradigmas testes dor independente examine processamento nociceptivo pelas duas grandes populações de fibras nervosas periféricas transmissão de dor, ou seja, as fibras não-mielinizadas C e pequenas mielinizadas A-delta fibras.

Determinar o calor e limiar de dor mecânica em pele inflamada de seres humanos

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

determining-heat-mechanical-pain-threshold-inflamed-skin-human

Research

JoVE Journal

Biology

20.7K visualizações.  Stanford University School of Medicine. Algoritmos de avaliar o limiar da dor de calor e mecânica em pele inflamada experimentalmente humana dos sujeitos do estudo são mostradas. Os dois paradigmas testes dor independente examine processamento nociceptivo pelas duas grandes populações de fibras nervosas periféricas transmissão de dor, ou seja, as fibras não-mielinizadas C e pequenas mielinizadas A-delta fibras.

Vídeo: Determining heat and mechanical pain threshold in inflamed skin of human subjects

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

Usando Micro-Electro-Mechanical Systems (MEMS) para desenvolver ferramentas de diagnóstico

Biologia

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

Transplante de pele murinos

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Implantação de engenharia de tecidos no coração de ratos

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

Demonstração de Allodynia Cutânea Em associação com dor pélvica crônica

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

Digital em linha Holographic Microscopy (Dihm) de Assuntos Fracamente de dispersão

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Imagem confocal de Solteiro mitocondriais superóxido Pisca em coração intacto ou<em&gt; In Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Ex vivo Cultura do Rato Embryonic pele e Live-imaging de Migração melanoblasto

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap juncional intercelular comunicação: um biomarcador funcional para avaliar os efeitos adversos de tóxicos e toxinas, e Benefícios para a saúde de produtos naturais

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Here we present a method to measure organismal fat levels in the third instar (L3) larval stage of Drosophila melanogaster. This method exploits the comparatively low density of fat tissue to differentiate between larvae with altered fat stores. Buoyancy-based analysis is a valuable tool for rapid, reproducible, and economical screening.

Um método baseado em flutuabilidade da determinação dos níveis de gordura no<em&gt; Drosophila</em

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat ...