German Research Foundation (FOR2240 "(Lymph) Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" to CC and LMH; HE 6743/2-1 and HE 7643/3-1 to LMH; CU47/6-1 to CC), German Cancer Aid (to LMH and CC), GEROK program University of Cologne (to SLS and LMH), and EU COST BM1302 "Joining Forces to Corneal Regeneration" (to CC).

O uso de tecidos humanos devem ser revistos e aprovados por um conselho de revisão institucional ou equivalente. O trabalho aqui descrito foi aprovado pelo comitê de ética local e teve a aprovação para análise científica. Este trabalho foi realizado de acordo com a Declaração de Helsinki. Os espécimes esclerais humanos foram obtidos a partir dos olhos de doadores globo (máximo de post-mortem de tempo de 24 horas), no Banco de Olhos do Departamento de Oftalmologia da Universidade de Colônia, Alemanha. 1. Preparação Experimental <ol><li> Preparar 96% de etanol e salino tamponado com fosfato (PBS) em diferentes tubos. Prepare os anticorpos primários na diluição recomendada de PBS contendo 2% de albumina de soro bovino (BSA) e manter todas as soluções em gelo. Preparação de 100 ul por amostra. </li><li> Limpe todos os instrumentos e usar bisturis afiados. Depois de repetido o mesmo corte com bisturi, alterar o bisturi. </li><li> Obter 1-2 forceps Colibri, micro tesouras de dissecação em linha reta, um bisturi # 10ou bisturi oftálmico micro pena, e quatro 26 agulhas G. </li><li> Enrole a folha de alumínio em torno de uma placa de poliestireno ou usar uma placa de cortiça para fixar o tecido. Trabalhar debaixo de um microscópio estéreo binocular. Trabalhar estéril debaixo de um fluxo de ar laminar, se necessário. </li></ol> 2. Prepare a esclera <ol><li> Pressione cuidadosamente a lâmpada e executar uma trepanação perfurante por parte córneo, e gire o trepan. Gire o trepan cuidadosamente e uniformemente sobre a superfície para realizar um corte igualmente rodada. Use um trepan tamanho 15,5 mm. Cortar os acessórios restantes com tesoura curva. Remova a trepanação córneo. Isto irá resultar em uma lâmpada anteriormente aberto. <ol><li> Coloque a esclera em um cotonete com a parte aberta para cima. Remova a retina e úvea, usando uma pinça colibri de retirar as duas camadas (retina e Uvea) da esclera. Não deixe a úvea pigmentada na esclera interior. Use tesoura curva para remover a retina e da úvea papila. Remover o REMaining conjuntiva, músculos extra-oculares, e da cápsula de Tenon da esclera superficial. </li></ol></li><li> Retirar a amostra escleral no tamanho necessário utilizando-tipo reto tesouras e fórceps Colibri. Tome cerca de 2 cm2 amostras esclerais porte de diferentes locais. Por causa do tamanho pequeno, segurar a esclera suavemente e cortar o tamanho exigido, quadrados usando os do tipo reta tesoura. A trepanação córneo define a margem anterior das amostras esclerais. <ol><li> Evitar repetido agarrando com a pinça como a área onde o tecido foi agarrado não é adequado para análise de microscopia confocal. Prepara-se o número necessário de amostras, dependendo do tipo de experiência (por exemplo, vs anteriormente posteriormente, comparar Figura 1) e a quantidade de anticorpos utilizada. </li></ol></li><li> Coloque as amostras em tubos de 1,5 ml para uso posterior. Fixar as amostras em 1,5 ml de etanol a 96% durante 15 minutos, e, em seguida, lavá-los três vezes cada durante 5 minem 1,5 ml de PBS no agitador. Se trabalhar com tecido congelado, execute essa etapa antes pressão congelamento. </li><li> Use tecido fresco. No entanto, se isso não for possível, pressão congelar a esclera em azoto líquido e mantê-lo a -20 ° C para uso posterior. Se trabalhar com tecido congelado, descongelar antes de usar. Evite o descongelamento repetido e ciclos de congelamento. </li><li> Manter cada amostra em PBS contendo 1,5 ml de BSA a 5% à temperatura ambiente durante 2 h. Este passo induz um aumento de volume da amostra, que é útil para a laminação e também previne a ligação inespecífica. </li><li> Depois que as amostras têm aumentado, preparar as praças esclerais para laminação. Trabalhar sob um microscópio, por exemplo, um microscópio estéreo binocular. Apor as amostras esclerais posteriores à membrana de poliestireno embrulhados em papel alumínio ou a placa de cortiça utilizando os 26 g de agulhas. </li><li> Dobrar as extremidades das agulhas, de forma que não interfiram com o campo visual sob o microscópio. </li><li> Laminar o SAMP escleral de espessura totalles. <ol><li> Primeiro, mantenha a borda escleral anterior usando uma pinça Colibri. Em seguida, cuidadosamente cortar uma fina camada superficial da esclera usando o bisturi # 10, o bisturi oftálmica micro pena, ou uma lâmina de uma espátula. Segurar o bisturi horizontalmente e cortar a camada superficial tão fina quanto possível, a partir da camada subjacente. </li><li> Dissecar uma camada fina escleral da praça escleral. Este método é semelhante às técnicas cirúrgicas convencionais para preparar uma aba durante trabeculectomia e deve resultar em camadas de espessura de 30-80 ^ m (comparar com a figura 2). </li></ol></li><li> Evitar a secagem da esclera pela adição de pequenas quantidades de PBS para o tecido, por exemplo, 50 ul de PBS utilizando uma pipeta. </li><li> Coloque as camadas cortadas em 100 ul de PBS na placa de 96 poços e rotular tanto a orientação (no sentido externo e interno) e a camada (superficial e profunda), por exemplo com uma cor à prova de água. </li><li> Repita os passos 2,7-2,9 até que o tecido está completamente laminado. &lt;/ Li&gt; </ol> 3. Execute Imunohistoquímica <ol><li> Adicionar 100 ul de anticorpos primários necessários para a diluição recomendada. Aqui, por exemplo, utilização de CD 31 LYVE1 anticorpo (anti-humano de coelho) (monoclonal de ratinho anti-humano) e, tanto em uma diluição de 1: 100 em PBS (contendo 2% de BSA) e incubar a 4 ° C durante a noite. Para alterar a forma da placa de 96 poços, segurar a pipeta para a parede do poço e remover o fluido. </li><li> No dia seguinte, as amostras de lavar três vezes cada durante 5 min com 200-300 ul de PBS no agitador. Adicionar 100 uL de anticorpos secundários correspondentes; aqui usar de cabra anti-rato fluoresceína isotiocianato (FITC) anti-coelho de cabra e corantes de cianina 3 (Cy3), e incuba-se as amostras à temperatura ambiente durante 1-2 horas. Dilui-se a anticorpos secundários 1: 300 em PBS contendo soro de cabra a 2%. </li><li> Lavar as amostras novamente três vezes cada durante 5 minutos em 200-300 ul de PBS no agitador. </li><li> Realização da coloração núcleo, por exemplo, </em&gt;. 4 &#39;, 6-diamidino-2-fenilindole (DAPI) (diluição 1: 2.000, 100 ul em cada poço e incubar 10 min à temperatura ambiente), depois lavar novamente 2 vezes em 200-300 ul de PBS no agitador. </li><li> Transferir as amostras em lâminas de microscópio, incorporá-los em 1-2 gotas de meio de montagem fluorescente, adicionar uma lamela, selá-lo, cobrindo as bordas com verniz transparente, e armazenar a 4 ° C para uso posterior ou examinar diretamente os slides com a confocal microscópio. </li><li> Usar um microscópio confocal (ou equivalente) para analisar as amostras. Use a ampliação desejada, por exemplo 10-40X ampliação. </li><li> Para verificar a espessura das amostras, medida com microscopia confocal usando Z-pilhas. A quantidade de possíveis secções esclerais depende da localização da esclerótica, tal como a espessura difere equatorial e posterior (comparar introdução). </li></ol>

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The authors declare no financial disclosures. The contents of this article are solely the responsibility of the authors.

Laminar a esclerótica humana é um método para a realização de microscopia confocal sobre este tecido. Um passo essencial neste processo é a utilização de etanol em vez de formalina para a aposição do tecido. Em nossa experiência, os melhores resultados são obtidos quando se utiliza etanol em vez de formol para fixação. bisturis Blunt agravar o processo e devem ser evitadas. Da mesma forma, a secagem da esclera deve ser evitado, uma vez que complica o processo e reduz a qualidade das imagens. <p class="...

A esclerótica é a camada externa rígida que cobre o olho, o que é feito de tecido conjuntivo denso. Isso ajuda a proteger as estruturas intra-oculares e para manter a pressão intra-ocular. Assim, a esclerótica é essencial para a visão clara. É desprovida de vasos linfáticos 1,2 e, assim, forma uma borda livre-linfática exterior entre ele e o olho interior livre de linfático 3-7. Ele também fornece locais de ligação para os músculos extra-oculares, partilhando assim semelhanças anatômicas com tendões. Porque a esclera consiste principalmente de feixes densos de colágeno tipo I e tem um número menor de colágeno tipo III, IV, V, VI, VIII 8,9 e elastina 10,11, este tecido não é fácil de usar para imuno-histoquímica. Anatomicamente, a esclerótica podem ser separadas em três camadas principais: (1) o episclera vascularizado superficial, localizado por debaixo da conjuntiva e da cápsula de Tenon e para os lados e the parte de trás do olho de frente para a órbita; (2) o estroma escleral, a parte principal da esclera; e (3) o fusca lâmina, que é uma camada fina, pigmentada localizada directamente acima da úvea. Nosso conhecimento anatômico sobre a esclera decorre, principalmente, da primeira metade do século 20. Naquela época, os investigadores estudaram a anatomia da vasculatura principalmente usando Índia injeções de tinta 12 e vascular lançando 13-15. Mais tarde, foi pesquisado em estudos angiográficos 16-19. Desde então, técnicas mais antigas foram melhoradas e novos foram desenvolvidos que nos permitiram completar conhecimento anatômico anterior. Por exemplo, tem sido apenas cerca de uma década desde que nós tivemos tais marcadores linfáticos confiáveis ​​como endotélio vascular linfática hyaluronan específica receptor-1 (LYVE1) 20 ou podoplanina 21. A microscopia confocal oferece novas possibilidades para o estudo das características anatômicas dos diferentes tissues do olho. Ele permite várias manchas a ser utilizada para diferenciar os marcadores de células ou para a localização das células em relação aos vasos sanguíneos e outras estruturas anatómicas. Ele fornece uma visão geral, quando a amostra é de um tamanho maior e permite-nos verificar através de uma amostra, quando em busca de um tipo de célula específico. Com a tecnologia Z-Pilha, microscopia confocal pode ser utilizado para as amostras até 100-200 um. A esclerótica difere de espessura entre 0,3 mm por trás das inserções musculares e 1 mm no pólo posterior 11. Devido tanto a sua espessura e opacidade, a esclera não é adequado para microscopia confocal utilizando métodos tradicionais. Para remediar esta situação, tecidos esclerais foram estratificados para permitir a sua análise com microscopia confocal. Esta tecnologia é útil para obter uma melhor compreensão de ambas as situações fisiológicas e patológicas na esclera humana.

<table>
	<tbody>
		<tr>
			<td>96% ethanol</td>
			<td>Merck Chemicals, Darmstadt, Germany</td>
			<td>P075.4</td>
			<td></td>
		</tr>
		<tr>
			<td>binocular stereo microscope&#160;</td>
			<td>Motic, Hongkong, China</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles&#160;</td>
			<td>Terumo, Leuven, Belgium</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr>
			<td>15.5mm trepan</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>no.10 scalpel&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td>2E+08</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic scalpel micro feather&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td colspan="2">no. 7657BR</td>
		</tr>
		<tr>
			<td>CD 31 antibody (monoclonal mouse anti human)</td>
			<td>Dako, USA</td>
			<td>IR610</td>
			<td></td>
		</tr>
		<tr>
			<td>LYVE1 antibody&#160; (polyclonal rabbit anti human)</td>
			<td>Zytomed, Germany</td>
			<td colspan="2">RBK014-05</td>
		</tr>
		<tr>
			<td>goat anti mouse FITC antibody</td>
			<td>Sigma Aldrich, Steinheim, Germany</td>
			<td>F0257</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rabbit Cy3 antibody</td>
			<td>Dianova, Germany</td>
			<td colspan="2">111-165-003</td>
		</tr>
		<tr>
			<td>Goat Serum normal</td>
			<td>Dako, Glostrup, Denmark</td>
			<td colspan="2">X090710-8</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Carl Roth GmbH, Karlsruhe, Germany</td>
			<td>6335.1</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides&#160;</td>
			<td>Engelbrecht, Ederm&#252;nde, Germany</td>
			<td colspan="2">WC7695002</td>
		</tr>
		<tr>
			<td>Coverslips 24x24mm</td>
			<td>Th. Gayer, Lohmar, Germany</td>
			<td>7695026</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO fluorescent mounting medium&#160;</td>
			<td>DAKO, USA</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM Meta 510 confocal microscopy&#160;</td>
			<td>Carl Zeiss AG, Jena, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a laminating technique to perform confocal microscopy of the human sclera

A esclerótica é um tecido conjuntivo denso que cobre e protege o olho. Ela consiste principalmente de feixes de colágeno densas (tipos I, III, IV, V, VI e VII). Devido à sua autofluorescência, opacidade, e espessura, não foi considerado adequado para a microscopia confocal. Uma abordagem alternativa para o apresentado aqui, que utiliza esclera fixado com formalina embebido em parafina para imuno-histoquímica, tem desafios técnicos, especialmente quando o pré-aquecimento do tecido para a recuperação de antigénio. Desde a esclera é relativamente pobre em ambas as células e vasos, o uso de amostras de tecido maiores foi explorada para ajudar a evitar que as células com vista e para compreender a sua localização em relação aos navios e outros sítios anatômicos. Para permitir a análise de amostras de tecido maiores, sob o microscópio confocal, uma técnica de laminação foi efectuada para criar camadas finas da esclera. Após a análise dos resultados dos vasos sanguíneos CD31 e vasos linfáticos endotelial hyalureceptor Ronan 1 (LYVE1) células positivas, para o qual foi obtido uma autorização para exame científico, as vantagens e limitações deste método são discutidas.

Nas experiências representativas realizadas aqui, há benefícios demonstráveis ​​derivados da utilização desta técnica de laminação em particular. A primeira experiência ilustra a rede diversificada de plexo de vasos sanguíneos episcleral em três imagens representativas (Figura 3). Os vasos são positivas para CD31. O segundo experimento mostra células do sistema imunológico, em particular de ...

Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

Usando uma técnica de estratificação para executar microscopia confocal da esclera humana


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

using-laminating-technique-to-perform-confocal-microscopy-human

Research

JoVE Journal

Medicine

7.5K visualizações.  University of Cologne. Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used. 

Vídeo: Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

Microscopia Confocal de córnea: uma técnica não-invasiva Novel para Quantificar Pequenas Fibre Patologia em neuropatias periféricas

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

Medicina

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Utilizando Estimulação Magnética Transcraniana para o Estudo do sistema neuromuscular humano

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

Sonda baseada Endomicroscopy Laser Confocal do Trato Urinário: A Técnica

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Procedimentos de biópsia de músculo esquelético humano, utilizando a técnica Bergström Modificado

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Construção de vapor Chambers usada para expor Ratos ao álcool durante o equivalente a todos os três trimestres de Desenvolvimento Humano

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rosa Bengala Fototrombose por imageamento óptico confocal<em&gt; In Vivo</em&gt;: Um Modelo de curso Veículo Individual

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

Desenvolvimento de um algoritmo para realizar um estudo abrangente de Disreflexia Autonômica em animais com alta Spinal Cord Injury Usando um dispositivo de telemetria

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

Tópica das vias aéreas Anestesia para intubação acordada-endoscópica Usando o Spray-as-you-go Técnica com fluxo de oxigênio elevado

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Segunda geração harmônica sinais na esclera coelho como uma ferramenta para avaliação do tecido terapêutico do cross-linking (TXL) para miopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Usando uma técnica de estratificação para executar microscopia confocal da esclera humana

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