Dr. Khan reconhece apoio de concessão da K12HD052892 NIH/FORMULADORES, a Alpha-1 Foundation, Fundação Hillman e centro de pesquisa do fígado de Pittsburgh. Dr. Michalopoulos reconhece apoio de concessão do NIH/NIDDK P01DK096990. Agradecemos a assistência técnica excelente e generosa de Anne Orr. Nós também estamos gratos ao Dr. David A. Geller para fornecer acesso ao laboratório de microcirurgia do transplante/roedor.

Todos os procedimentos foram realizados em conformidade com as diretrizes éticas do Comité de uso da Universidade de Pittsburgh e institucional Cuidado Animal.
1. básicas técnicas e procedimentos comuns
<ol><li>Autoclave todo equipamento microcirúrgico antes do uso.</li>
	<li>Para evitar matar o rato pela anestesia, verificar cuidadosamente seu padrão de respiração.</li>
	<li>Órgãos de toque e intestinos o mínimo possível para evitar danificar os órgãos e causando sangramento. Pressione pontos de sangramento com cotonetes de algodão molhado ou esponjas de gaze.</li>
	<li>Use cotonetes de algodão molhado para manipulação suave dos órgãos. Esponjas de gaze molhada, não-tecidos são utilizadas em dois tamanhos, 3.5 × 3,5 cm e 6,0 × 6,0 cm. usam o menorzinho para retrair o fígado inteiro. Use o maior para embrulhar os intestinos pequenos e grandes.</li>
</ol>2. pré-operatória preparação
<ol><li>Use Camundongos c57/b6 do sexo masculino, 8-12 semanas de idade (quando o peso do corpo é aproximadamente 20-35 g) para ligadura parcial de ducto biliar.</li>
	<li>Permitir que os ratos livre acesso à água e comida até a indução da anestesia.</li>
</ol>3. parcial biliar ligadura operação
<ol><li>Anestesia o mouse, colocando o animal em uma câmara de indução com uma mistura de 1,5-2,0% de isoflurano e 1 L/min de oxigênio. Monitore o animal cuidadosamente observando-se a taxa de respiração. Quando a taxa de respiração do animal é uma respiração por segundo, o rato é bem anestesiado. Desde que o reflexo de retirada do membro dianteiro desaparece mais rapidamente, aperte o pé do membro posterior também para confirmar a ausência completa do reflexo de retirada. Os olhos devem ser lubrificados com um agente lubrificante oftálmico para evitar úlceras da córnea. Nota a primeira dose de analgésico pode ser dado pré-operatório para prevenir a estimulação dos receptores de dor.</li>
	<li>Raspar a parede abdominal com máquina de cortar cabelo e coloque o mouse em cima da mesa cirúrgica sob o microscópio cirúrgico. Corrigi todos os membros para a mesa com a fita. Use a inalação de isoflurano para manter o rato sob anestesia geral.<ol>
<li>Ajustar o vaporizador de isoflurano e usar isoflurano 2.0-3.0% para indução.</li>
	</ol>
</li>
	<li>Usando cotonetes estéreis, desinfectar a parede abdominal primeiro com 0,3-0,5 mL de iodo-povidona aplicado topicamente, seguido por 70% de etanol. Animal deve ser coberto com um pano estéril.</li>
	<li>Fazer uma longa abdominal incisão do xifoide até o púbis, usando a tesoura e pinça Adson. Depois de fazer a incisão abdominal, reduzir isoflurano 0.8-1.0% para manutenção.</li>
	<li>Expor na cavidade abdominal por meio de um Microafastador. Segure o xifoide com pinça mosquito e retrair-se em direção a cabeça do rato. Fixar a pinça mosquito com argila macia.</li>
	<li>Instilar 1 a 2 mL de aquecida (37 ° C) esterilizado fosfato salino (PBS) na cavidade abdominal para evitar ferir os órgãos abdominais. Segure o intestino delgado com compressas de algodão molhado e retrair-os suavemente em direção a cabeça do rato. Corte os ligamentos entre o intestino delgado e o retroperitônio para evitar ferir o intestino delgado (figura 1A).</li>
	<li>Enrole os intestinos pequenos e grandes com uma gaze molhada, não-tecidos. Coloque-os no lado caudal da cavidade abdominal. Intestinos, envolvidos em gaze estéril podem ser colocados no Campo drapeado estéril para evitar contaminação.</li>
	<li>Gentilmente retrai o fígado inteiro em direção a xifoide usando um pequeno molhado não-tecido de gaze para expor o Hilo hepático e ligamento hepato-duodenal (figura 1B).</li>
	<li>Verificação para confirmar a anatomia do hilo hepático. O ducto biliar comum está localizado no lado ventral da veia portal. Confirme os locais do direito e o ducto biliar hepático esquerdo, que drenam para a confluência biliar principal no hilo hepático.</li>
	<li>Identificar o ducto biliar hepático esquerdo que drena o lobo esquerdo e cercá-lo com uma sutura de nylon 10-0 usando o lado sem corte de uma agulha de sutura (Figura 2A). Evite ferir a superfície hepática, artéria hepática e veia porta.</li>
	<li>Ligate do ducto biliar esquerdo com nylon 10-0. (Figura 2B).</li>
	<li>Coloque os intestinos pequenos e grandes na mesma posição que antes da operação, usando cotonetes de algodão molhado.</li>
	<li>Confirmar que não há nenhuma torção intestinal e hemorragia abdominal e, em seguida, feche o músculo e a pele em duas camadas com vicryl 4-0. Sutura trançada (Vicryl) foi usada para fechamento da pele, mas é preferível monofilamento para reduzir a probabilidade de infecção. Nota: Feche a fáscia por sutura contínua do xifoide ao púbis e fechar a pele por sutura contínua do púbis ao xifoide.</li>
	<li>Pare de anestesia depois de fechar a incisão.</li>
</ol>4. acompanhamento e tratamento pós-operatório
<ol><li>Só após a cirurgia, beliscar a pele dorsal do mouse e injetar Buprenex (0,1 mg/kg) e cefazolina (100 mg/kg) por via subcutânea, seguido por Buprenex injeção cada 12 h por 3 dias e cefazolina cada 24 h por 3 dias respectivamente11,12 ,13. Nota a primeira dose de analgésico pode ser dado pré-operatório para prevenir a estimulação dos receptores de dor. Cefazolina e outros antibióticos de peri-operatório são necessários apenas para animais imunitário suprimido, como aqueles usados em estudos de transplante. Nota: Se a cirurgia é realizada com êxito, o mouse geralmente se recupera dentro de 30 min. actividade dos ratos será quase o mesmo que a condição pré-operatória.</li>
	<li>Verifique os ratos diariamente por sinais de aflição.<ol>
<li>Sacrifica os ratos pelo deslocamento cervical sob anestesia geral para adquirir fígados e outras amostras de tecido em pontos de tempo definido (isto é, 7, 14, 21 dias) depois de pBDL.</li>
	</ol>
</li>
</ol>

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Os autores têm sem divulgações.

Embora cBDL é a mais comum técnica experimental para colestase obstrutiva, pode apresentar vários desafios em roedores. Dependendo do número de dias pós-operatórios necessários para alcançar um ponto de extremidade, os animais podem experimentar significativa morbidade e mortalidade que o tempo avança. O grau de lesão tecidual e necrose biliar é ainda agravado quando se trabalha com alguns modelos de rato transgénico de doença hepática, que demonstram o parênquima hepática anormal e função na linha de b...

Modelos cirúrgicos da lesão hepática aguda e reparação, tais como hepatectomy parcial ou ligadura do ducto biliar comum (cBDL), têm sido utilizados há décadas em roedores para estudar a regeneração hepática e Patobiológico1,2. Em cBDL, é ligado o ducto biliar comum (que drena todas bile produzida no fígado), resultando em colestase obstrutiva, inflamação e fibrose nas primeiras 2-3 semanas após a cirurgia, com eventual progressão para cirrose3. Proliferação do ducto biliar, transdiferenciação celular e indução de células progenitoras de fígado residente são também características da cBDL4,5. Embora cBDL elucida mecanismos específicos de cholangiopathy obstrutiva, que são bem caracterizados com resultados reprodutíveis em ratos do selvagem-tipo e nessas cepas com fígados normal6,7,8, o procedimento pode ser tecnicamente exigente em alguns modelos de rato transgénico, doenças do fígado, que exigem mais animais do que o previsto devido à maior morbidade e mortalidade de obstrução biliar, ao longo do tempo. Esta técnica é também benéfica para estudar doença de ratos transgénicos com subjacentes fígado, que não pode tolerar senão o stress das experiências de curso prolongado após cBDL.
pBDL pode fornecer uma alternativa razoável para ajudar a superar alguns desses desafios. Estudos de pBDL inicial em ratos do selvagem-tipo visa distinguir localmente atuante e sistêmicos de mediadores da inflamação9. De pBDL, confluência biliar principal de direita e esquerdas hepáticas biliares acima o ducto biliar comum com cuidado é visualizada no hilo, e apenas esquerdo hepático ducto biliar é ligado para gerar uma colestase obstrutiva localizada. pBDL oferece várias vantagens para cBDL. De pBDL, unligated lóbulo direito serve como um controle interno forma idêntica correspondente para o lobo esquerdo ligado, sujeitado para os mesmos efeitos sistêmicos, tais como o estado nutricional ou fatores de circulação. Embora mediadores pró-inflamatórios sistêmicos estavam presentes, Osawa et al observou menos fibrose e necrose dos lóbulos unligated, mas aumento de células inflamatórias e expressão de crescimento fator-β transformador do lóbulo ligados9.
Mais recentemente, foi redefinido para estudar efeitos parenquimatosos hepáticas da bile retida10pBDL. Usando o mesmo animal para controle interno (unligated "sham") e grupos experimentais, pBDL metades efetivamente o número de animais necessários por experiência, que por sua vez, é mais rentável. Os ratos toleram os efeitos de pBDL muito melhor, para que o tempo ideal de curso experimentar (≥2 semanas) podem ser alcançadas no lóbulo ligado. Mais importante ainda, pBDL pode distinguir efeitos directos de colestase da colestase obstrutiva e retidos componentes da bile no lóbulo ligado, em comparação com o lobo de unligated controle. Em geral, pBDL é um método atrativo para estudar efeitos específicos do lóbulo da colestase localizada no fígado.

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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Microscope-Leica Wild M650, 6&#8211;40&#215; magnification</td>
			<td style="width:336px;">Leica Microsystems</td>
			<td style="width:195px;">n/a</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tec 3 isoflurane funnel fill vaporizer</td>
			<td>General Anesthetic Services</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stevens Tenotomy Scissors</td>
			<td>Accurate Surgical &#38; Scientific Instruments Corporation</td>
			<td>ASSI.9193</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps</td>
			<td>Fine science tools</td>
			<td>11006-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mosquito classic delicate hemostatic forceps</td>
			<td>Codman</td>
			<td>30-4472</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-retractor</td>
			<td>Murdock; Roboz Surgical Instrument</td>
			<td>RS-6550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonwoven gauze sponges</td>
			<td>Fisherband</td>
			<td>870-PC-DBL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puritan 6&#8221; Small Cotton Swab w/Wooden Handle</td>
			<td>Puritan Medical Products Company</td>
			<td>868-WCS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont SS Forceps - Standard Tips/Straight/Inox/13.5cm</td>
			<td>Fine science tools</td>
			<td>11203-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont SS Forceps - Standard Tips/Angled 45&#176;/Inox/13.5cm</td>
			<td>Fine science tools</td>
			<td>11203-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Spring Scissors - 3mm Cutting Edge</td>
			<td>Fine science tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microneedle holder</td>
			<td>Aesculap Surgical Instruments</td>
			<td>FD231R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro AROSuture, sterile 10-0 nylon suture, 70 &#181;m, TAP points</td>
			<td>AROSurgical Instruments Corporation</td>
			<td>T4A10N07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl</td>
			<td>Ethicon</td>
			<td>J662H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-ml Syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon tissue culture dish, 60 &#215; 15 mm</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:343px;">Isoflurane, United States Pharmacopeia (USP) liquid for inhalation, 250 ml</td>
			<td>Piramal Healthcare</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenex injectable (buprenorphine hydrochloride)</td>
			<td>Reckitt Benckiser Pharmaceuticals</td>
			<td>NDC 12496-0757-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefazolin for injection , USP</td>
			<td>APP Pharmaceuticals</td>
			<td>NDC 63323-238-61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVP scrub solution (povidone&#8211;iodine 7.5%)</td>
			<td>Medline</td>
			<td>NDC 12496-0757-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Phosphate Buffered Saline Without Calcium or Magnesium</td>
			<td style="width:336px;">LONZA</td>
			<td style="width:195px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sirius Red</td>
			<td style="width:336px;">Sigma</td>
			<td style="width:195px;">365548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Hematolxylin</td>
			<td style="width:336px;">Fisher (Richard-Allan Scientific)</td>
			<td style="width:195px;">22-050-111 (7211)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Eosin</td>
			<td style="width:336px;">Anatech (Fisher)</td>
			<td style="width:195px;">832 (NC9686037)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Formalin</td>
			<td style="width:336px;">Fisher</td>
			<td style="width:195px;">SF100-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

partial bile duct ligation in the mouse: a controlled model of localized obstructive cholestasis

Em roedores, ligadura do colédoco completa (cBDL) do ducto biliar comum é uma técnica cirúrgica estabelecida para estudar a proliferação de colestase e ducto biliar obstrutiva. No entanto, as experiências a longo prazo podem levar a aumento da morbidade e mortalidade. Em cepas do mouse selecione com doença hepática subjacente, uma comparação significativa pode ser feita mesmo com ligadura de um único lóbulo do fígado, que pode reduzir as despesas e perdas de animais. Aqui, descrevemos a ligadura parcial de ducto biliar (pBDL) no rato, no qual é ligado somente à esquerda hepático ducto biliar, causando obstrução biliar no lobo esquerdo, mas não os lóbulos remanescentes. Com cuidado técnica microcirúrgica, experimentos de pBDL podem ser rentáveis, desde que o lobo unligated serve como um controle interno para os lóbulos ligados, quando submetidos às mesmas condições no mesmo animal. Ao contrário da cBDL, um grupo de controle de operação separado não é necessário. pBDL é altamente útil diretamente comparar localizada contra efeitos sistêmicos de colestase e outros componentes da bile retida. pBDL também pode ser reaproveitado como um novo método para investigar os mecanismos relacionados a medicamentos e a migração celular.

Para este experimento típico, pBDL foi realizado em ratos transgénicos triplicados PiZ, que recapitular a doença do fígado humana encontrada na deficiência de alfa-1 antitripsina (A1AT). O rato de PiZ overexpresses um transgene consiste em múltiplos fragmentos genomic do DNA que contêm as regiões codificação do gene humano mutante de A1AT e promotor, em conjunto com os intrões e ~ 2 kilobases de upstream e downstream flanqueiam regiões14, <s...

Watch this Scientific Journal Video about Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis at JoVE.com

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

Aqui, apresentamos a ligadura parcial biliar como modelo cirúrgico de lesão hepática e regeneração em roedores.

Ligadura do colédoco parcial no Mouse: um modelo controlado da colestase obstrutiva localizada

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

partial-bile-duct-ligation-mouse-controlled-model-localized

Research

JoVE Journal

Biology

15.4K visualizações.  Jichi Medical University. Aqui, apresentamos a ligadura parcial biliar como modelo cirúrgico de lesão hepática e regeneração em roedores.

Vídeo: Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

O Probe microfluídicos: Operação e Uso de Processamento de Superfície localizada

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Biologia

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

LAD Ligadura: Um Modelo murino de Infarto do Miocárdio

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

Indução cirúrgica da hidropisia endolinfática por obliteração do ducto endolinfático

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualização de mRNAs retículo endoplasmático localizados em células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Injeção Intraductal para localizada Drug Delivery ao Rato glândula mamária

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

A validação de um modelo de camundongo para Disrupt Complexos LINC de um modo específico de célula

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Um modelo do rato de obstrução Intestinal parcial

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Isolamento e cultura sanduíche de colágeno 3D de hepatócitos de camundongos primários para estudar o papel do citoesqueleto na formação bile canalicular in vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...