A fonte de financiamento para a demonstração laboratório estava concessão NIGMS: GM077119. Agradecemos ao Dr. Joseph R. Haywood e Dr. Peter Cobbett pelo apoio do Short Couse em Integrative e Órgão Sistemas de Farmacologia. Agradecemos também a Senhora Hannah Garver por seu apoio técnico da demonstração laboratorial.

Antes de qualquer procedimento animal, o cuidado com os animais e uso comitê institucional (IACUC) deve aprovar o protocolo. Este protocolo foi aprovado pela Michigan State University IACUC. 1. Preparação de pré-laboratório de solução de FITC-inulina <ol><li> Aqueça 20 ml de solução salina a 70 ° C e agita-se lentamente em 100 mg de FITC-inulina (5 mg / ml de FITC-inulina) de inulina é até que todo dissolvido. </li><li> Solução arrefecer até à TA e adicionar 800 mg de albumina de soro de bovino (40 mg / ml de BSA, em pó liofilizada, essencialmente livre globulina, baixa endotoxina, ≥98% de pureza por electroforese em gel de agarose). </li><li> Filtra-se a solução de inulina-BSA com papel de filtro (grau 1). Coloque a solução filtrada em uma seringa de 20 ml com um filtro de seringa com ponta (0,2 um) e cobrir com folha de a proteger da luz. </li></ol> 2. Anestesia e Cirurgia <ol><li> Coloque o rato em uma câmara de indução preenchido com 5% de isoflurano para induzir a anestesia. Grave bodpeso y (250-350 g) e colocar o rato numa plataforma cirúrgica aquecido concebido para manter 37 ° C a temperatura do corpo durante todo o experimento. Segurar cuidadosamente o rato para a plataforma com fita de laboratório sobre as patas. Manter a anestesia com 1-2% de isoflurano com classe médica 100% O 2 a taxa de fluxo de ar de 0,8-1,0 L / min. </li><li> Inserir um cateter cônico (OD ponta intravascular, 2.7F) na artéria femoral para pressão arterial e monitoramento da freqüência cardíaca, e coleta de sangue. </li><li> Inserir um cateter (PE-50) na veia femoral para infusão de inulina. Prenda o cateter ao tecido com 5-O trançado de seda de sutura cirúrgica 6 circundante. </li><li> Fixe o cateter arterial a um transdutor de pressão calibre de tensão. Pressão arterial e freqüência cardíaca registro usando o software de aquisição de dados e exibição em uma tela de computador em tempo real. Esta técnica é demonstrado em detalhe em vídeo 6. </li><li> Expor a bexiga através de uma incisão suprapúbica. Corte um pequenoorifício na ponta da bexiga e da inserção de uma cânula (PE-190) com uma ponta de calor queimado no interior da bexiga para a recolha de urina. Fixe a cânula para a bexiga com uma sutura em bolsa. </li></ol> 3. urina e coleta de sangue <ol><li> Colocar a seringa de FITC-inulina em uma bomba de seringa com velocidade de fluxo definido de 1 ml / h por 100 g de peso corporal (3 ml / h durante um rato pesando 300 g). Fixe a seringa para o cateter na veia femoral. Iniciar a infusão de inulina e permitir um período de equilíbrio de 1-2 horas. Manter seringa coberto de uma camada para proteger da luz. </li><li> Determinar se o ritmo de fluxo de urina é estável e adequado para a análise de amostras (20 ul / min) através da recolha de uma amostra de urina de uma pré-pesados ​​para um frasco de recolha período de 10 min. Determinar o volume de urina por gravimetria com uma balança digital. Um volume de urina suficiente durante um período de recolha de 10 min é de 0,2 ml. Continuar a recolher amostras de urina até duas coleções consecutivos indicam uma taxa de fluxo de urina de 20 l / mou mais. </li><li> Amostras pré-droga <ol><li> Recolher uma amostra de urina durante um período de 20 min. Recolher uma amostra de sangue (0,5 ml) a partir do cateter arterial, no ponto médio do período de recolha de urina. Tenha o cuidado de limpar completamente o cateter arterial de solução salina antes de coletar uma amostra de sangue em um frasco contendo uma coleção U heparina. Use frascos de coleta com marcações de volume para facilitar a recolha de 0,5 ml de sangue arterial. </li><li> Lave o catéter arterial com heparina-solução salina (20 U / mL) para limpar o cateter de sangue (aprox. 0,1 mL). O comprimento do cateter arterial deve ser tão curto quanto possível, para limitar o volume de solução salina de heparina-necessária para descarregar. Nota: as amostras de sangue diluído produzir cálculos imprecisos de TFG e excreção fracionada de Na e K. </li><li> Aguarde 10 minutos e repita a cobrança de uma segunda urina Pré-drogas e amostra de sangue. </li></ol></li><li> Após a coleta de duas amostras de pré-droga, administrar um diurético drug, a furosemida (10 mg / kg), através do cateter arterial. Lave o cateter arterial com solução salina heparinizada para limpar o cateter de drogas. Ter o cuidado de evitar a injecção de ar através do cateter arterial. Anote o tempo da injeção de furosemida. </li><li> As amostras post-droga: Em cada um dos três pontos de tempo a seguir, recolher uma amostra de urina durante um período de recolha de 10 min, e uma amostra de sangue (0,5 ml) no ponto médio do período de recolha de urina. <ol><li> Para Sample Post-Drogas 1 - coletar cinco minutos após a furosemida. </li><li> Para Post-Drogas Amostra 2 - recolher dez minutos após a furosemida. </li><li> Para Sample Post-Drogas 3 - recolher quinze minutos após a furosemida. </li></ol></li><li> Depois de todas as amostras foram coletadas, eutanásia do rato em conformidade com os procedimentos institucionais por toracotomia e remoção do coração. Remova ambos os rins. Decapsulate (remover a membrana que envolve) e apagar os rins para remover o excesso de sangue. Pesar os rins. </li></ol><ol><li> Meça todos os volumes de amostras de urina por gravimetria com uma balança digital, e pesos recordes. </li><li> Amostras de sangue total de centrifugação, utilizando uma centrífuga de bancada (1800 x g) para separar o plasma. Transferir as amostras de plasma para pequenos frascos rotulados. </li><li> Analisar as concentrações de sódio e potássio em amostras de urina e plasma com um analisador de sódio / potássio. </li><li> Medição de FITC-inulina no plasma e urina <ol><li> Dilui-se a urina de pré-droga (de 1: 200 a 1: 400), e na urina pós-fármaco (1:10) com tampão HEPES (500 mM, pH 7,4). </li><li> Adicionar 40 ul de padrão ou de amostra ul e 60 de tampão HEPES numa placa de 96 cavidades (uma amostra por cavidade) e deixa-se misturar durante 10 min enquanto se coberto com folha de alumínio. </li><li> Gerar uma curva padrão para FITC-inulina, relativamente às concentrações de 6,25, 12,5, 25, 50, 100, 200, 400 ug / ml (Figura 1). Determinar a fluorescência de FITC-inulina em amostras e padrões usando um leitor de microplacas com excitação e comprimentos de onda de emissão de 485 e 538 nm, respectivamente. </li><li> Encaixe os valores fluorescentes para os padrões de uma análise de regressão função logística de 4 paramter. Os parâmetros da função de regressão são usados ​​para calcular a concentração de FITC-inulina em amostras de plasma e de urina (Tabela 1). </li></ol></li></ol> 5. Pós-laboratório Análise de Resultados: Cálculos <ol><li> Calcular Urina Taxa de Fluxo (UV; ml / min): [volume de urina recolhida (ml)] ÷ [tempo da recolha (min)] </li><li> Calcular Taxa de Filtração Glomerular (GFR; ml / min): [a concentração de inulina urina (ug / ml) x UV (ml / min)] ÷ [inulina conc plasma. (Ug / ml)] </li><li> Calcular filtrada Carga de sódio (pmol / min): concentração de sódio no plasma (nmol / ml) x TFG (ml / min) </li><li> Calcular excreção de sódio Classificação (U Na V; umol / min): concentração de sódio na urina (pmol / ml) x UV (ml / min) </li><li> Calcule Fractional excreção de sódio (FE Na;%): [L Na V (pmol / min)] ÷ [Carga de sódio filtrado (pmol / min)] x 100 </li><li> Calcular carga filtrada potássio (pmol / min): a concentração de potássio no plasma (nmol / ml) x TFG (ml / min) </li><li> Calcular excreção de potássio Classificação (L K V; umol / min): concentração de potássio da urina (pmol / ml) x UV (ml / min) </li><li> Calcule Fractional excreção de potássio (K FE;%): [U K V (mmol / min)] ÷ [Load potássio filtrado (mmol / min)] x 100 </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes. As opiniões ou afirmações contidas neste documento são os pontos de vista particulares do autor e não devem ser interpretadas como funcionário ou como refletindo os pontos de vista do Departamento do Exército ou do Departamento de Defesa.

Um marcador apropriado para a medição de GFR deve atender quatro critérios: ser livremente filtrada no glomérulo, ser independente para proteínas do plasma, e nem ser absorvido nem secretada no néfron. A inulina é um polímero de frutose, que satisfaz estes critérios. Como resultado, a depuração renal da inulina é considerada o padrão ouro para medir GFR 7. A técnica demonstrada representa a abordagem tradicional da determinação da eliminação renal de inulina utilizando recolhas de urina cron...

The most important function of the kidney is the homeostatic regulation of extracellular water and electrolyte content. The kidneys closely regulate extracellular water, sodium (Na) and potassium (K) to maintain normal physiological levels. Disturbances in renal function can result in serious metabolic disorders which can be fatal. The basic renal process occurs in the nephron and begins with the filtration of plasma at the glomerulus and ends with the excretion of urine. Other processes that determine the final concentration of water, Na and K in the urine are secretion and reabsorption within the nephron. Measurements of glomerular filtration rate (GFR) and the fractional excretion of Na and K are critical in assessing renal function in health and disease. The reader is referred to previously published review articles and textbooks for a more thorough discussion of kidney function1-4. 
GFR can be measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron5. While this technique requires anesthesia, surgical preparation, and a terminal experiment, it is considered the gold standard of GFR measurement. Using inulin that is tagged with fluorescein-isothiocyanate (FITC), plasma and urine concentration of FITC-inulin can be easily measured in small volumes and used to calculate GFR during multiple time points of an experiment. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. 
The conceptual understanding of how to measure renal function can easily be demonstrated in a short lab designed to allow students to actively participate in some aspects of the experiment. This video depicts the pre-lab preparation, the renal function demonstration, and the post-lab evaluation of results. The surgical techniques necessary for making measurements of GFR are demonstrated in an anesthetized rat. In addition, example calculations for GFR, and the fractional excretion of Na and K are shown before and after administration of a diuretic drug.

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		<tr>
			<td>5-0 Braided Silk Surgical Suture</td>
			<td>Surgical Specialties Corp</td>
			<td>SP1033</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Plate, 96-Well</td>
			<td>Costar&#160;</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Chemical Co</td>
			<td>A2934-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>MicroFuge 18, 357160</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Sample Tubes</td>
			<td>Dot Scientific Inc.&#160;</td>
			<td>#711-FTG</td>
			<td></td>
		</tr>
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			<td>Cotton Tipped Applicators</td>
			<td>Solon Manufacturing Co</td>
			<td>56200</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition Software</td>
			<td>ADInstruments</td>
			<td>LabChart Pro 7.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Scale&#160;</td>
			<td>Denver Instrument</td>
			<td>APX-4001</td>
			<td></td>
		</tr>
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			<td>FITC-Inulin</td>
			<td>Sigma Chemical Co</td>
			<td>F3272-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Sponges</td>
			<td>Covidien</td>
			<td>2146</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated Surgical Bed</td>
			<td>EZ-Anesthesia</td>
			<td>EZ-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagnet</td>
			<td>NDC 25021-402-10</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Chemical Co</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott Animal Health</td>
			<td>IsoFlo, 5260-04-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Vaporizer</td>
			<td>EZ-Anesthesia</td>
			<td>EZ-190F</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Forceps</td>
			<td>Biomedical Research Instruments Inc.</td>
			<td>70-1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader - Fluoroskan</td>
			<td>ThermoScientific</td>
			<td>Ascent FL, 5210460</td>
			<td></td>
		</tr>
		<tr>
			<td>NOVA 5+ Sodium/Potassium Analyzer</td>
			<td>NOVA BioMedical</td>
			<td>14156</td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holders with Scissors</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td></td>
		</tr>
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			<td>PE-190 (for bladder catheter)</td>
			<td>BD Medical</td>
			<td>427435</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Transducer&#160;</td>
			<td>ADInstruments</td>
			<td>MLT1199</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex Culture Tubes</td>
			<td>Corning Inc.</td>
			<td>99445-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Femoral Tapered Artery Catheter</td>
			<td>Strategic Applications Inc.</td>
			<td>RFA-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Salix Furosemide 5%</td>
			<td>Intervet</td>
			<td>#34-478</td>
			<td></td>
		</tr>
		<tr>
			<td>Strabismus Scissors</td>
			<td>Fine Science Tools</td>
			<td>14075-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>91402-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Gloves</td>
			<td>Kimberly-Clark</td>
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			<td>George Tiemann&#160; Co</td>
			<td>105-420</td>
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</table>
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physiology lab demonstration: glomerular filtration rate in a rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

O diurético usado no laboratório demonstração foi furosemida que muito rapidamente inibe a reabsorção de Na e K filtrada pelo rim, resultando em aumento de Na, K, e a excreção de água dentro de minutos após a administração da droga. Por seu mecanismo primário, furosemida deve ter efeitos mínimos sobre a TFG e da carga filtrada de Na e K, mas vai aumentar o fluxo de urina, e fração de excreção de Na e K. Os resultados representativos da Tabela 3 mostram que,...

Watch this Scientific Journal Video about Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat at JoVE.com

Demonstração de laboratório de fisiologia: taxa de filtração glomerular em um rato | Biologia | Vídeo

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

Laboratório de Fisiologia Demonstração: Taxa de filtração glomerular em um Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

physiology-lab-demonstration-glomerular-filtration-rate-in-a-rat

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17.2K visualizações.  U.S. Army Institute of Surgical Research. The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function. 

Vídeo: Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

Volume grande (20L +) Filtração das amostras Seawater Coastal

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

Biologia

Small Volume (1-3L) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

Volume pequenas (1-3L) Filtragem de Amostras de Água do Mar Coastal

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O&#x2019;Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and S&#233;guinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."

Although there is considerable evidence that the hippocampus is involved in piloting (O&#x2019;Keefe and Nadel, 1978; O&#x2019;Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O&#x2019;Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria&#x2013;fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.

In a piloting scent tracking task, the ability of the rats to return to a refuge with food using visual an odor trail or using dead reckoning in infrared light, the integrated record of previous movements, demonstrates that the hippocampus is necessary for dead reckoning.

Uma demonstração em vídeo de Pilotagem Preservado por Rastreamento Scent mas Dead Reckoning alterado após a fímbria-Fornix Lesões no Rat

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

Ver histórico e demonstração Fisiologia da MNJ da Muscle Opener Crayfish

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

Ovariectomia e 17β-estradiol substituição em ratos e camundongos: uma demonstração visual

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Um Método de alta capacidade para medição de Taxa de filtração glomerular em ratos conscientes

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Rápido isolamento e purificação de mitocôndrias para o transplante pela dissociação do tecido e Filtração Diferencial

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Uma Experimental e Bioinformática Protocolo de Análises de RNA-seq de fotoperiódicas diapausa no Tigre Asiático Mosquito,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Usando o rato do mamárias Células Tumorais para ensinar Biologia Conceitos Básicos: A Módulo Lab Simples

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperf Técnica para Investigar Regulamento de microvasos Permeabilidade de Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...