Reconhecemos a assistência hábil de Annie B. Kristensen, Elisabeth V. Andersen, Belinda B. Bringtoft e os cuidadores de animais no Departamento de Medicina Experimental, Frederiksberg Campus da Faculdade de Saúde e Ciências Médicas da Universidade de Copenhague, na Dinamarca. Jacob Lønholdt está agradeceu por sua assistência técnica excelente em filmar os procedimentos. Este estudo foi financiado em parte pelo Centro LIFEPHARM de farmacologia in vivo e da Universidade de Copenhagen.

Os procedimentos mostrados foram realizados de acordo com protocolos aprovados pelo animal dinamarquês Experimentação Inspecção no âmbito do Ministério da Alimentação, Agricultura e Pescas. 1. Sangue Amostragem do lateral safena ou Tarsal Veia <ol><li> Execute este procedimento sem anestesia e com a ajuda de um investigador, uma manipulação a cobaia, e uma coleta de amostra. </li><li> Raspar a região tarsal da perna com um barbeador elétrico até que as veias são visíveis. Certifique-se de que a área é grande o suficiente para evitar interferir com a pele no local de punção. </li><li> Coloque a perna em um banho de água morna e esfregue suavemente a perna, fazendo com que as veias mais dilatada e evidente. </li><li> Limpe a perna com uma toalha limpa e desinfectar o local da punção com gaze ou um chumaço de algodão umedecido com álcool 70%. </li><li> Contenha a cobaia e estender a perna para baixo, aplicando pressão suave imediatamente acima do joelho to fornecer estase. </li><li> Por punção da veia com uma agulha de 21 G ou lanceta. Recolha de sangue, por exemplo., Usando um tubo de recolha ou capilar. Gentilmente manipular a perna para ajudar o fluxo de sangue. </li><li> Solte estase, uma vez que a amostra foi obtida. Aplique um pedaço de gaze ou algodão, com uma leve pressão ao local da punção até que o sangramento parou. </li><li> Retorne o animal para sua gaiola e monitor por 5-10 min para assegurar a hemostasia. </li></ol> 2. coleta de sangue da veia da orelha <ol><li> Executar este procedimento, sem anestesia, quer isoladamente ou, de preferência com a ajuda de um colega. </li><li> Desinfetar a superfície dorsal da orelha com gaze ou um chumaço de algodão umedecido com álcool 70%. </li><li> Aplicar uma leve pressão para a base da orelha para proporcionar estase. </li><li> Por punção da veia com uma agulha de 21 G ou lanceta, e coleta de sangue. </li><li> Solte estase, uma vez que a amostra foi obtida, aplique um pedaço de gaze ou algodão com gepressão ntle ao local da punção até que o sangramento parou. </li><li> Retorne o animal para sua gaiola e monitor por 5-10 min para assegurar a hemostasia. </li></ol> 3. Sangue Amostragem da veia jugular <ol><li> Realizar este procedimento com a ajuda de um investigador adicional; uma manipulação da cobaia e uma coleta de amostra. </li><li> Anestesiar a cobaia com um anestésico adequado durante toda a duração deste procedimento. Uma mistura de tiletamina (0,93 mg / kg), zolazepam (0,93 mg / kg), xilazina (1,49 mg / kg) e butorfanol (0,06 mg / kg) pode ser usado. </li><li> Coloque a cobaia anestesiada em suas costas e retirar delicadamente as pernas dianteiras caudal. </li><li> Raspar e desinfectar (por ex., Com 70% de álcool) a parte ventral do pescoço. </li><li> Cubra a seringa com heparina ou EDTA antes da amostragem, a fim de reduzir o risco de coagulação. </li><li> Palpar a clavícula e inserir um 25 G (ou 23 G) agulha montada em um 1 ml ap seringaximadamente 1 cm lateral à linha média ao nível da articulação do ombro. Manter a seringa em um pequeno ângulo para cima e medial enquanto aplica uma pressão ligeiramente negativa. Evite danificar as estruturas subjacentes como a veia jugular está situado muito superficial. Inclinação do gargalo da cobaia ligeiramente para trás, para proporcionar o aumento da estase venosa. </li><li> Libertar as pernas da frente, quando a amostra de sangue é obtida, e aliviar a estase antes de a agulha é retraída. Use um pedaço de gaze seca ou algodão para aplicar pressão suave para o local da punção. </li><li> Retorne o animal para uma gaiola de recuperação e monitoramento para garantir a hemostasia e recuperação da anestesia. </li></ol>

<ol>
	<li>DeOgburn, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+increased+dietary+cholesterol+with+carbohydrate+restriction+on+hepatic+lipid+metabolism+in+Guinea+pigs.">Effects of increased dietary cholesterol with carbohydrate restriction on hepatic lipid metabolism in Guinea pigs.</a> Comp.Med. 62 (2), 109-115 (2012).</li><li>Fernandez, M., Volek, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guinea+pigs+a+suitable+animal+model+to+study+lipoprotein+metabolism,+atherosclerosis+and+inflammation.">Guinea pigs a suitable animal model to study lipoprotein metabolism, atherosclerosis and inflammation.</a> Nutr Metab (Lond). 3 (17), (2006).</li><li>Frikke-Schmidt, H., Tveden-Nyborg, P., Birck, M. M., Lykkesfeldt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+dietary+fat+and+cholesterol+exacerbates+chronic+vitamin+C+deficiency+in+guinea+pigs.">High dietary fat and cholesterol exacerbates chronic vitamin C deficiency in guinea pigs.</a> Br J Nutr. 105 (1), 54-61 (2011).</li><li>Hem, A., Smith, A. J., Solberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saphenous+vein+puncture+for+blood+sampling+of+the+mouse,+rat,+hamster,+gerbil,+guinea+pig,+ferret+and+mink.">Saphenous vein puncture for blood sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret and mink.</a> Lab Anim. 32 (4), 364-368 (1998).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> J Pharmacol Pharmacother. 1 (2), 87-93 (2010).</li><li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+guidelines+and+recommendations.">FELASA guidelines and recommendations.</a> J Am Assoc Lab Anim Sci. 51 (3), 311-321 (2012).</li><li>Ye, P., Cheah, I. K., Halliwell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+fat+diets+and+pathology+in+the+guinea+pig+Atherosclerosis+or+liver+damage.">High fat diets and pathology in the guinea pig Atherosclerosis or liver damage.</a> Biochim Biophys Acta. 1832 (2), 355-364 (2013).</li><li>Tveden-Nyborg, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+vitamin+C+deficiency+during+pregnancy+persistently+impairs+hippocampal+neurogenesis+in+offspring+of+guinea+pigs.">Maternal vitamin C deficiency during pregnancy persistently impairs hippocampal neurogenesis in offspring of guinea pigs.</a> PLoS One. 7 (10), 12-18391 (2012).</li><li>Schjoldager, J. G., Tveden-Nyborg, P., Lykkesfeldt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+maternal+vitamin+C+deficiency+overrides+preferential+fetal+ascorbate+transport+but+does+not+influence+perinatal+survival+in+guinea+pigs.">Prolonged maternal vitamin C deficiency overrides preferential fetal ascorbate transport but does not influence perinatal survival in guinea pigs.</a> Br J Nutr. 110 (9), 1573-1579 (2013).</li><li>Greulich, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+products+of+guinea+pig+epicardial+fat+induce+insulin+resistance+and+impair+primary+adult+rat+cardiomyocyte+function.">Secretory products of guinea pig epicardial fat induce insulin resistance and impair primary adult rat cardiomyocyte function.</a> J Cell Mol Med. 15 (11), 2399-2410 (2011).</li><li>Swifka, J., Weiss, J., Addicks, K., Eckel, J., Rosen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+fat+from+guinea+pig+a+model+to+study+the+paracrine+network+of+interactions+between+epicardial+fat+and+myocardium.">Epicardial fat from guinea pig a model to study the paracrine network of interactions between epicardial fat and myocardium.</a> Cardiovasc Drugs Ther. 22 (2), 107-114 (2008).</li><li>Pilny, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+hematology+of+rodent+species.">Clinical hematology of rodent species.</a> Vet.Clin North Am Exot Anim Pract. 11 (3), 523-vii (2008).</li><li>Franco, N. H., Olsson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientists+and+the+3Rs:+attitudes+to+animal+use+in+biomedical+research+and+the+effect+of+mandatory+training+in+laboratory+animal+science.">Scientists and the 3Rs: attitudes to animal use in biomedical research and the effect of mandatory training in laboratory animal science.</a> Lab Anim. 48 (1), 50-60 (2014).</li><li>Van, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=eAssessing+the+application+of+the+3Rs+a+survey+among+animal+welfare+officers+in+The+Netherlands.">eAssessing the application of the 3Rs a survey among animal welfare officers in The Netherlands.</a> Lab Anim. 47 (3), 210-219 (2013).</li><li>Sloan, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+doses+of+ketamine-xylazine+anesthesia+reduce+cardiac+ischemia-reperfusion+injury+in+guinea+pigs.">High doses of ketamine-xylazine anesthesia reduce cardiac ischemia-reperfusion injury in guinea pigs.</a> J Am Assoc Lab Anim Sci. 50 (3), 349-354 (2011).</li><li>Jacobson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+anaesthetic+regimen+for+surgical+procedures+in+guinea+pigs.">A novel anaesthetic regimen for surgical procedures in guinea pigs.</a> Lab Anim. 35 (3), 271-276 (2001).</li><li>Saha, J. K., Xia, J., Grondin, J. M., Engle, S. K., Jakubowski, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+hyperglycemia+induced+by+ketamine+xylazine+anesthesia+in+rats+mechanisms+and+implications+for+preclinical+models.">Acute hyperglycemia induced by ketamine xylazine anesthesia in rats mechanisms and implications for preclinical models.</a> Exp Biol Med(Maywood). 230 (10), 777-784 (2005).</li></ol>

Os autores não têm nada a revelar. Todos os procedimentos foram aprovados pela Lei de Experimentação Animal da Dinamarca, que está de acordo com o Conselho da Europa Convenção ETS 123.

Em comparação com camundongos e ratos, cobaias estão com medo, por natureza e deve ser abordada com calma e tratada corretamente, a fim de reduzir o estresse. Do ponto de vista do bem-estar animal, agindo com cuidado é naturalmente uma prioridade; no entanto, este também irá reduzir o risco de efeitos associados a ansiedade sobre o por exemplo, dados coletados., aumento dos níveis sanguíneos de corticosteróides e glicose 12. Da mesma forma, a movimentação diária irá habituar os animais c...

A cobaia um modelo experimental animal valioso e validado devido a um número de semelhanças biológicos para os seres humanos, tais como a exigência de uma fonte dietética de vitamina C, a lipoproteína plasmática comparável enzimas que metabolizam e perfis de lipoproteína, bem como semelhanças compartilhadas com placentação humana e desenvolvimento pré-natal 1-3,7,8. Isso faz com que a cobaia um modelo atraente e adequado para estudar os efeitos de doenças associadas à dieta, como a doença não alcoólica do fígado gorduroso, doenças cardiovasculares e efeitos putativos de deficiência de vitamina C, mas também o efeito de, por exemplo., A intervenção dietética materna sobre o desenvolvimento prole. Coleta de sangue repetida muitas vezes é necessária tanto em curto e longo prazo em estudos in vivo. Devido às diferenças anatómicas específicas de espécies é necessário modulação da técnica de amostragem, a fim de obter amostras de sangue em cobaias, em comparação com outros modelos de roedores <sup&gt; 4,5. Isto inclui a variação ao longo de veias e artérias, e também que as cobaias não tem uma cauda, ​​assim, ter diferentes requisitos e opções de amostragem e de manuseamento. Amostragem do safena ou veia do tarso, induz o mínimo de desconforto e estresse no animal e não requer anestesia 3,9. Esta técnica permite que as amostras de pequenas quantidades de sangue (100-400 ul), por exemplo, repetido., Para determinar os marcadores bioquímicos no plasma. Se um tamanho ainda mais pequeno da amostra é suficiente (50-100 ul), este pode facilmente ser obtido por punção da veia da orelha. Esta técnica também pode ser levada a cabo em animais conscientes e é útil, por exemplo, quando a medição da glucose no sangue 10,11. A amostragem a partir da veia jugular permite a recolha de maiores quantidades de sangue (1-2 ml), mas requer anestesia. Aqui, apresentamos quatro técnicas de amostragem de sangue diferentes para ambos cobaias conscientes ou anestesiados. O procedures são todos os procedimentos não-terminal na condição de os volumes de amostra e número de amostras não exceder as diretrizes para coleta de sangue em animais de laboratório 6. É no interesse da boa ciência, bem como de bem-estar animal que o estresse deve ser reduzido ao mínimo. Consequentemente, apenas bem treinados e pessoal competente deve executar os procedimentos com animais.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:295px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:315px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Electric shaver</td>
			<td style="width:295px;">Wella (Contura), Denmark</td>
			<td style="width:119px;">273207</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Syringes&#160;</td>
			<td style="width:295px;">B.Braun-Omnifix-F, Germany</td>
			<td style="width:119px;">9161406V</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Needles</td>
			<td style="width:295px;">BD Microlance,Spain</td>
			<td style="width:119px;">301156</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microvette collection tube&#160;</td>
			<td style="width:295px;">Sarstedt, D-51588N&#252;mbrecht, Germany</td>
			<td style="width:119px;">201,288,100</td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Accu-Check analyzer</td>
			<td style="width:295px;">Roche A/S Diagnostics, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Heparin 5000 IE/ml</td>
			<td style="width:295px;">LEO Pharma, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Zoletil 50, Chemvet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Narcoxyl vet 20 mg/ml, Intervet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Anesthesia</td>
			<td style="width:295px;">Torbugesic vet 10 mg/ml, Scanvet, Denmark</td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Neutral eye ointment</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gauze or a cotton swab&#160;</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disinfectant (70% alcohol)</td>
			<td style="width:295px;"></td>
			<td style="width:119px;"></td>
			<td>Alternative product may be used. Multiple suppliers</td>
		</tr>
	</tbody>
</table>

non-terminal blood sampling techniques in guinea pigs

As cobaias possuem várias semelhanças biológicas para os seres humanos e são validados modelos animais experimentais 1-3. No entanto, o uso de cobaias representa atualmente uma área relativamente estreita de dados de pesquisa e descritivos sobre metodologia específica é correspondentemente escasso. As características anatômicas de cobaias são um pouco diferentes de outros modelos de roedores, daí modulação de técnicas de amostragem para acomodar as diferenças espécie-específicos, por exemplo., Em comparação com camundongos e ratos, são necessários para obter amostras suficientes e de alta qualidade. Como tanto a longo e curto prazo em estudos in vivo, muitas vezes requerem repetidas sangue amostragem a escolha da técnica deve ser bem considerado, a fim de reduzir o estresse e desconforto nos animais, mas também para garantir a sobrevivência, bem como o cumprimento dos requisitos de tamanho da amostra e acessibilidade. Amostras de sangue venoso pode ser obtido em uma série de sites em cobaias por exemplo., A safena e jugularveias, cada uma das técnicas que contém tanto vantagens como desvantagens 4,5. Aqui, apresentamos quatro técnicas de amostragem de sangue diferentes para ambos cobaias conscientes ou anestesiados. Os procedimentos são todos os procedimentos não-terminal na condição de os volumes de amostra e número de amostras não exceder as diretrizes para coleta de sangue em animais de laboratório 6. Todos os métodos descritos têm sido exaustivamente testado e aplicado para repetido em amostras de sangue vivo em estudos dentro do nosso centro de pesquisa.

Quatro abordagens diferentes para in vivo de técnicas de amostragem de sangue não-terminal, nem em cobaias conscientes ou anestesiados foram apresentados. Figura 1 ilustra o curso da safena lateral e veia tarsal na cobaia e possíveis locais de punção. Veias da orelha visíveis na superfície dorsal de uma orelha da cobaia são ilustrados na Figura 2 Estas veias pode ser utilizado para a amostragem de sangue, quando apenas um volume muito pequeno, é necessária. A posiçã...

Watch this Scientific Journal Video about Non-Terminal Blood Sampling Techniques in Guinea Pigs at JoVE.com

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Apesar de ser um modelo conhecido, a cobaia representa atualmente um nicho em ciências experimentais em animais e dados limitada disponível sobre a execução da maioria dos procedimentos. Apresentamos aqui quatro abordagens diferentes para não-terminal em técnicas de amostragem de sangue vivo em qualquer cobaias conscientes ou anestesiados.

Técnicas de coleta de sangue não terminal em cobaias

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

non-terminal-blood-sampling-techniques-in-guinea-pigs

Research

JoVE Journal

Biology

37.3K visualizações.  University of Copenhagen.  Apesar de ser um modelo conhecido, a cobaia representa atualmente um nicho em ciências experimentais em animais e dados limitada disponível sobre a execução da maioria dos procedimentos. Apresentamos aqui quatro abordagens diferentes para não-terminal em técnicas de amostragem de sangue vivo em qualquer cobaias conscientes ou anestesiados. 

Vídeo: Non-Terminal Blood Sampling Techniques in Guinea Pigs

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

A amostragem da água do mar e Cobrança

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Biologia

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

A gravação óptica de atividade elétrica na Guiné-porco Networks Enteric usando Tensão sensível Corantes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Técnicas de imagem Ca 2 + Sinalização no esperma humano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Grampo patch e Técnicas de Perfusão para estudar canais iônicos Expresso em Xenopus Oócitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Técnicas de Análise de vesículas extracelulares utilizando citometria de fluxo

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.

The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.

Manchas de sangue seco - preparação e processamento para uso em imunoensaios e em técnicas moleculares

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

A amostragem de sangue a partir da veia lateral da cauda do rato

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

Técnicas de fenotipagem mícron-escala de milho feixes vasculares, com base na tomografia computadorizada de Microcomputed de raio-x

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

Procedimentos de Gonadectomia e Amostragem de Sangue no Modelo teleost de pequeno tamanho Japonês Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...