We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1. Tissue Colheita e congelamento dos tecidos musculares <ol><li> Eutanásia o rato com uma overdose de isoflurano (2-cloro-2- (difluorometoxi) -1,1,1-trifluoro-etano). Execute esta etapa em um exaustor e usar um método de contenção secundária, como um secador ou uma redoma de vidro contendo um cotonete embebido em isoflurano. </li><li> Confirme a morte com firmeza apertando pata. Use um método secundário de eutanásia, como luxação cervical. </li><li> Molhar a pele com álcool a 70% para reduzir a aderência de fios de cabelo soltos sobre os músculos. Descasque a pele fora o membro usando uma pinça fina, tais como # 5. </li><li> Cuidadosamente separar o músculo de interesse (tibial anterior (TA), neste caso) a partir dos ossos por baixo, a partir do tendão e remover. </li><li> Extirpar o músculo, cobri-lo em outubro (de corte ideal composto de temperatura), e coloque-o deitado sobre um molde de congelamento descartável rotulados. Certifique-se de que o músculo está em sua orientação fisiológica normal (cabeça para tail) sem esticar. Alternativamente, congelar o músculo em pequenos quadrados de isopor e fixá-lo utilizando os pinos de insetos para garantir o comprimento correto dos músculos. </li><li> Relaxar 2-metilbutano (isopentano) em um copo de aço usando azoto líquido. Isso leva, em média, 3-5 min. Continue mexendo de forma intermitente até precipitados brancos começam a se formar no fundo e paredes do copo. NOTA: Quando isso ocorre, isopentano tenha alcançado a temperatura ideal de congelamento (-150 ° C) </li><li> Mergulhe os moldes contendo músculos na gelada de 2-metilbutano. Congelar pequenos músculos, como o diafragma e longus extensor digitorum (EDL) para 6-12 seg e músculos maiores, como o sóleo gastrocnêmio (GS) e tríceps para 15 - 20 seg. Não congelar os músculos por muito tempo, pois isso pode levar a fissuras. </li><li> Transfira os tecidos congelados sobre gelo seco e deixe evaporar isopentano (note que este processo leva em média cerca de 15 - 20 min). Enrole os tecidos em papel alumínio e armazená-los em ultra baixa temperatura (-70 ° C a -80 ° C) até o corte. </li></ol> 2. Incorporar os tecidos em outubro de Elaborar bloco de tecido <ol><li> Tecidos de transporte em gelo seco para cyrostat para evitar o descongelamento. Transferência para o tecido criostato câmara (definida a -20 a -24 ° C para evitar o descongelamento completo de tecidos) e deixá-los equilibrar durante 30 min. </li><li> Vire a baía Peltier de refrigeração por diante. Se o cryostat não tem esse resfriamento, use aerosol spray de arrefecimento para congelar rapidamente a outubro Não deixe que o degelo do músculo em qualquer ponto durante a incorporação, pois isso pode resultar em danos causados ​​por congelamento. </li><li> Adicione uma camada fina e uniforme de outubro no disco de amostra e deixe esfriar. Quando a maior parte dos PTU é congelada no fundo enquanto ainda líquido na parte superior, insira o tecido na orientação correta (perpendicular outubro para seções transversais e paralelas para outubro para cortes longitudinais). Use spray de aerosol arrefecimento para congelar rapidamente outubro Toque na parte unembeded do tecido com calor extractor e aguarde 45 segundos. </li><li> Adicionar outra camada fina de outubro em torno do músculo, pulverizador com de aerossol de pulverização de arrefecimento e extrair o calor tal como no passo anterior. </li><li> Continua a adicionar outubro em pequenos incrementos e congelar rapidamente OCT utilizando uma combinação de aerossol de pulverização e extractor de calor de arrefecimento até que todo o músculo é coberto. </li><li> Colocar o extractor de calor na parte superior do bloco de tecido e esperar durante 5 min. </li></ol> 3. Preparar Seções e identificando as secções de Mid-barriga Região dos Músculos <ol><li> Montar o espécime para o cabeçote da amostra. Aperte o botão para fixar o disco. Certifique-se de que o disco de amostra está em bom contato com o cabeçote de amostra. </li><li> Ajuste a temperatura da câmara de entre -21 e -24 ° C e aguarde até que a temperatura definida é alcançada. </li><li> Ajuste as configurações de espessura (7 um); aparar o bloco através do avanço ou retração do bloco usando configurações grossas e finas. </li><li> Recolher as seçõesem pré-aquecido lâminas de microscópio com carga positiva, pressionando a parte superior da lâmina para as secções, utilizando a atracção entre cargas opostas para facilitar a aderência das secções de corte na lâmina. </li><li> Identificar as secções médio-barriga por medição da área da secção transversa (AST) das secções. Faça isso usando software de imagem no computador que está ligado ao microscópio. NOTA: As imagens de contraste de fase são tomadas e circunferência de todo o músculo é medido através de uma função de rastreamento sobre o programa de software. Isto irá proporcionar tanto CSA, bem como diâmetro mínimo furão (medida de fibras de tamanho em corte transversal que é expressa como a menor distância entre duas tangentes paralelas em lados opostos de uma partícula) medições. Nota: Quando CSA e diâmetro mínimo ferret medições começar a platô, mid barriga foi atingido. </li><li> Inventário e arquivar as lâminas a -80 ° C para a coloração futuro. NOTA: o PTU não precisam ser removidos antes da staininprocedimentos de g. Uma vez que as lâminas são lamínulas, eles estão prontos para ser manchado imediatamente. </li></ol> 4. Coloração NOTA: Embora os procedimentos passo a passo detalhadas podem ser encontradas na folha de dados do produto e deve ser seguido por procedimentos de coloração, otimização de pessoal pode ser obrigado a obter a coloração ideal. <ol><li> Como meios de montagem (ver quadro) não seja miscível com água, por meio de lâminas de desidratar graus crescentes de álcool (2 min cada em 70%, 95%, e 100% de etanol) e limpá-las com xileno (100% durante 5 min) para garantir lâminas não se tornam opaca ao longo do tempo. </li><li> Lamela seções e deixe-os secar. Imagem com microscópio equipado com software de imagem. </li></ol>

<ol>
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History, clinical status, muscle strength, and biopsy findings.</a> AMA J Dis Child. 91, 326-338 (1956).</li><li>Platzer, A. C., Chase, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+Alterations+in+Preclinical+Mouse+Muscular+Dystrophy.">Histologic Alterations in Preclinical Mouse Muscular Dystrophy.</a> The American journal of pathology. 44, 931-946 (1964).</li><li>Mercuri, E., 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=Muscle+magnetic+resonance+imaging+involvement+in+muscular+dystrophies+with+rigidity+of+the+spine.">Muscle magnetic resonance imaging involvement in muscular dystrophies with rigidity of the spine.</a> Ann Neurol. 67, 201-208 (2010).</li><li>Tasca, G., 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=Different+molecular+signatures+in+magnetic+resonance+imaging-staged+facioscapulohumeral+muscular+dystrophy+muscles.">Different molecular signatures in magnetic resonance imaging-staged facioscapulohumeral muscular dystrophy muscles.</a> PloS one. 7, e38779 (2012).</li><li>Girgenrath, M., Dominov, J. A., Kostek, C. A., Miller, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+apoptosis+improves+outcome+in+a+model+of+congenital+muscular+dystrophy.">Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy.</a> The Journal of clinical investigation. 114, 1635-1639 (2004).</li><li>Kumar, A., Yamauchi, J., Girgenrath, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle-specific+expression+of+insulin-like+growth+factor+1+improves+outcome+in+Lama2Dy-w+mice,+a+model+for+congenital+muscular+dystrophy+type+1A.">Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A.</a> Human molecular genetics. 20, 2333-2343 (2011).</li><li>Mehuron, T., 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=Dysregulation+of+matricellular+proteins+is+an+early+signature+of+pathology+in+laminin-deficient+muscular+dystrophy.">Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy.</a> Skeletal muscle. 4, 14 (2014).</li><li>Yamauchi, J., Kumar, A., Duarte, L., Mehuron, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggering+regeneration+and+tackling+apoptosis:+a+combinatorial+approach+to+treating+congenital+muscular+dystrophy+type+1+A.">Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A.</a> Human molecular genetics. 22, 4306-4317 (2013).</li><li>Sheriffs, I. N., Rampling, D., Smith, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+wax+embedded+muscle+is+suitable+for+the+diagnosis+of+muscular+dystrophy.">Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy.</a> Journal of clinical pathology. 54, 517-520 (2001).</li><li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+fixation.">Formaldehyde fixation.</a> Journal of Histochemistry &amp; Cytochemistry. 33, 845-853 (1985).</li><li>Maccarty, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Diagnostic+Reliability+of+Frozen+Sections.">The Diagnostic Reliability of Frozen Sections.</a> The American journal of pathology. 5, 377-380 (1929).</li><li>Shi, S. 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=Evaluation+of+the+value+of+frozen+tissue+section+used+as+'gold+standard'+for+immunohistochemistry.">Evaluation of the value of frozen tissue section used as 'gold standard' for immunohistochemistry.</a> Am J Clin Pathol. 129, 358-366 (2008).</li><li>Turan, T., 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=Accuracy+of+frozen+sections+for+intraoperative+diagnosis+of+complex+atypical+endometrial+hyperplasia.">Accuracy of frozen sections for intraoperative diagnosis of complex atypical endometrial hyperplasia.</a> Asian Pacific journal of cancer prevention : APJCP. 13, 1953-1956 (2012).</li></ol>

The authors have nothing to disclose.

A histologia e imuno-histoquímica têm sido utilizados como ferramentas fundamentais para a compreensão da manifestação e progressão da patologia de várias desordens neuromusculares. Classicamente, biópsias musculares foram fixadas pela primeira vez em formaldeído e depois incorporado com cera de parafina. Enquanto fixação em formol preserva a arquitetura do tecido e permite o armazenamento e transporte de tecido à temperatura ambiente, também inativa enzimas e proteínas ligações cruzadas que obrigariam a...

O exame de histologia muscular desempenha um papel crítico na compreensão de diferentes tipos de distúrbios neuromusculares 1-4. Quando combinado com outras técnicas, permite que pesquisadores para ter uma idéia melhor da manifestação ea progressão da patologia e também pode ser essencial para avaliar a eficácia de uma intervenção terapêutica 5-10. No entanto, para ser exato, os tecidos precisam ser preservados de forma adequada, de modo a preservar o estado fisiológico imediatamente antes da excisão. Isso requer muito cuidado durante a remoção, preservação, seccionamento e coloração. Os tecidos podem ser preparados de duas maneiras diferentes para estudo microscópico luz. O método primeiro, fixados em formalina embebidos em parafina (FFPE) seções, requer tecidos fixados em formol 10% tamponado naturais antes de ser incorporado com cera de parafina 11,12. A etapa de fixação ajuda a preservar a morfologia melhor, mas também ligações cruzadas pr endógenooteins necessitando, portanto, de antigénios intrincado procedimentos de recuperação para a imunocoloração e eliminando a possibilidade de coloração enzimática em tais seções 12. As secções congeladas, por outro lado, não precisa de ser pré-fixo ou processado; por conseguinte, as proteínas são capazes de reter conformações nativas biológicos 13,14. Além disso, cortes congelados também permitem rápido tempo de resposta, que às vezes é necessário, por exemplo, no diagnóstico intra-operatório de sarcomas e outras biópsias cirúrgicas 15. No entanto, se não for feito corretamente, este método é propenso a congelar danos, que introduz alterações à arquitetura muscular fazendo as seções impróprios para exame histológico. Este artigo irá delinear o método mais eficaz para preparar biópsias musculares, a fim de alcançar uma óptima coloração para exame adequado.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cryostat</td>
			<td>Leica CM 1850&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td>Buffalo Grove, IL 60089 United States, office phone 1-800 248 0123 
			Richard Allen Scientific</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse 50i&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Olympus</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Nikon Imaging Software Basic Research</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Image J, Metamorph</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>14043-220-05</td>
			<td>JD Medical Dist. Co., Inc.</td>
			<td>JD Medical Distrubuting Co., Inc 1923 West Peoria Avenue Phoenix, Arizona 85029</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>4583</td>
			<td>Sakura Inc.</td>
			<td>Sakrua Fintek, USA, Torrence, CA 90501, USA 
			Leica Microsystem</td>
		</tr>
		<tr>
			<td>Freeze It</td>
			<td>23-022524</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Disposable tissue mold</td>
			<td>22-363-554</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Colorfrost plus slides</td>
			<td>9991001</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>650501-4L</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>HC-1300-1GL</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>M 32631-SL</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>HC-7001GA</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Cytoseal 280</td>
			<td>8311-4</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Hematoxylin Gill #3</td>
			<td>CS402-1D</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>6766007</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
	</tbody>
</table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

A avaliação histológica de biópsias do músculo tem servido como uma ferramenta indispensável para a compreensão do desenvolvimento e progressão da patologia de doenças neuromusculares. No entanto, a fim de fazê-lo, o cuidado apropriado deve ser tomado quando excisão e preservar tecidos para alcançar a coloração ideal. Um método de preservação de tecido envolve a fixação tecidos em formaldeído e, em seguida, incorporando-os com cera de parafina. Este método preserva morfologia bem e permite o armazenamento a longo prazo a temperatura ambiente, mas é difícil e requer a manipulação de produtos químicos tóxicos. Além disso, os resultados de fixação em formaldeído antígeno cross-linking, o que exige protocolos de recuperação antigênica para immunostaining eficaz. Pelo contrário, o seccionamento congelado não necessita de fixação e, assim, retém a conformação antigénio biológica. Este método também fornece uma vantagem distinta em volta rápida em torno do tempo, tornando-se especialmente útil em situações que necessitam de avaliação histológica rápido como intraoperativa biópsias cirúrgicas. Aqui nós descrevemos o método mais eficaz de preparação de biópsias musculares para visualização com diferentes colorações histológicas e imunológicas.

A instalação para a colheita e congelamento de tecidos pode ser visto na Figura 1. Tecidos excisados ​​deve ser armazenado em uma gaze embebida em PBS para evitar a secagem (Figura 1A). Quando os tecidos são selecionados para serem utilizados para análise histológica eles devem ser mergulhados em outubro e dispostas em molde crio na orientação adequada e tão perto de comprimento fisiológico possível para garantir a morfologia preciso (Figura 1B). Polpa de ...

Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

Do&#039;s and Don&#039;ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Fazer e não fazer na preparação de músculo Cortes congelados para análise histológica

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

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Medicine

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

31.4K visualizações.  Boston University. Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Vídeo: Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

Em função Assay Canine Muscle Vivo

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Medicina

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

High-throughput Citometria de Fluxo Ensaio Celular para detectar anticorpos em N-metil-D-aspartato ou dopamina-2 Receptor em Soro Humano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Avaliação ultra-som base de Artéria Coronária Flow e Fluxo Coronariano Reserve Usando o modelo de sobrecarga de pressão no Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Humano<em&gt; vasto lateral</em&gt; Biópsia de músculo esquelético Usando o Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Dissecação do pâncreas Mouse para análise histológica e perfis metabólicos

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

Exame ecocardiográfico e histológico da morfologia cardíaca no Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

Avaliação In Vivo das Propriedades Mecanicas e Viscoelásticas da Língua do Rato

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Amostragem e Extração de Tumores Cerebrais no local para análise metabolômica e lipidômica

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

Técnicas de Dissecção e Amostragem Histológica do Coração em Modelos de Grandes Animais para Doenças Cardiovasculares

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

Preparação do tecido miocárdio humano para cultivo a longo prazo

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...