Agradecemos sinceramente à Fundação Whitehall por seu apoio ao financiamento e fundos de startup do Scripps Research Institute para a realização deste trabalho.

1. Preparação de Gânglios Abdominais, Medidas Eletrofisiológicas e Isolamento de Neurônios Identificados Únicos do Gânglio Abdominal da Aplysia californica
<ol><li>Mantenha a Aplysia no aquário de laboratório com água do mar artificial circulante (ASW) a 16 °C sob 12:12 claras:condições escuras.</li>
	<li>Isolamento do gânglio abdominal.<ol>
<li>Anestesiar animais injetando 380 mM MgCl2 solução por 5-10 min (equivalente a 30-35% do peso corporal do animal).</li>
		<li>Identificar o gânglio abdominal com base em sua posição no sistema nervoso central24,28.</li>
		<li>Remova o gânglio por operação cirúrgica e armazene imediatamente em água do mar artificial composta por 450 mM NaCl, 10 mM KCl, 10 mM CaCl2, 55 mM MgCl2, 2,5 mM NaHCO3e 10 mM HEPES (pH 7.4).</li>
	</ol>
</li>
	<li>Tratamento protease.<ol>
<li>Incubar o gânglio abdominal por 30 min a 34 °C±0,5 em uma placa de Petri com 0,1% de protease (dispase) diluída em ASW descrita acima. NOTA: A quantidade da protease utilizada precisa ser padronizada com a idade e o peso dos animais. Em geral, animais mais velhos e maiores exigiriam mais protease.</li>
	</ol>
</li>
	<li>Remoção da baia de gânglio.<ol>
<li>Após o tratamento enzimante, fixe o gânglio na base de silicone sylgard de uma câmara celular (Ø= 1 cm; vol. = 0,3 ml) e perfuse com ASW (taxa de fluxo: 150 μl/min) à temperatura ambiente (18 °C±2 ). NOTA: Para aumentar a visibilidade dos neurônios identificados, coloque o gânglio na parte superior de um cilindro de vidro de policarbonato(Figura 1) (diâmetro Ø= câmara celular modificada de 2 mm) que pode ser facilmente iluminado a partir da parte inferior usando um estereómico binóculo com ampliação de 20X e 40X.</li>
		<li>Remova cuidadosamente a bainha do gânglio usando fórceps e microscisores.</li>
	</ol>
</li>
	<li>Gravação eletrofisiológica simultânea de dois neurônios.<ol>
<li>Identifique neurônio de interesse.</li>
		<li>Empalear o neurônio com uma única microeletrância afiada intracelular com resistência de 10-15 MΩ preenchida com solução de 3 M KCl.</li>
		<li>Registre a atividade neuronal (passe de banda de 10 kHz) usando um sistema de gravação intracelular.</li>
		<li>Processe os dados de gravação usando softwares de eletrofisiologia, como o Axograph. NOTA: Pode-se facilmente realizar a gravação de múltiplos neurônios. Os neurônios L7, L11 ou R15 e R2 são facilmente identificados com base em sua posição. Dois amplificadores e dois micromanipuladores são necessários para o registro simultanea de dois neurônios L7 e R15(Figura 2).</li>
	</ol>
</li>
	<li>Aplicação de drogas.<ol>
<li>Aplique a droga de escolha na câmara celular por tubulação suave ou injete diretamente nos neurônios.</li>
		<li>Realizar medições eletrofisiológicas. NOTA: Dependendo do objetivo experimental, pode-se medir diferentes parâmetros eletrofisiológicos de neurônios, como as alterações no potencial de membrana (MP) ou potencial de ação (AP) antes, durante e depois da aplicação da droga.</li>
	</ol>
</li>
	<li>Isolamento de neurônios únicos.<ol>
<li>Ao final dos experimentos eletrofisiológicos, pare a perfusão do ASW e lave o gânglio com 100% de etanol.  A exposição ao etanol petrificará os neurônios e, usando fórceps finos, facilitará a remoção de neurônios individuais sem danificar outros neurônios do gânglio.</li>
		<li>Remova neurônios únicos sob um estereótipo e transfira para um pequeno tubo Eppendorf contendo reagente de extração de RNA gelado de 500 μl. Neurônios únicos isolados podem ser armazenados no reagente de extração de RNA a -80 °C para análise futura.</li>
	</ol>
</li>
</ol>2. Isolamento de RNA de neurônios únicos e análise de expressão genética por PCR em tempo real quantitativo (qPCR)
<ol><li>Precauções para minimizar a degradação do RNA: As ribonucleases (RNases) degradam o RNA e são enzimas muito estáveis e ativas. Tome as seguintes etapas durante o manuseio de RNAs.<ol>
<li>Limpe a área de trabalho e os instrumentos com agentes desativadores da RNase.</li>
		<li>Use luvas o tempo todo enquanto manuseia RNA e troque as luvas com frequência.</li>
		<li>Use apenas plásticos livres RNase para lidar com RNAs.</li>
	</ol></li>
	<li>Isolando o RNA total de neurônios únicos:<ol>
<li>O protocolo Trizol padrão para isolamento de RNA pode ser usado para isolar RNAs de neurônios únicos. Adicione brevemente 20% v/v de clorofórmio ao Trizol, misture bem por um breve vórtice.</li>
		<li>Coloque os tubos no rotador por 10 min a 4 °C. Gire a mistura Trizol-Cloroforme a 12.000 x g por 15 min a 4 °C.</li>
		<li>Pegue a fase aquosa e transfira para um tubo fresco.</li>
		<li>Adicione a solução de acetato de sódio (pH 5.5) a uma concentração final de 0,3 M, 100 ng/ml do coprecipitante GlycoBlue, e 2,5 volumes de 100% de etanol para precipitar RNA.</li>
		<li>Misture bem as amostras e incubar a -80 °C durante a noite.</li>
		<li>Gire as amostras a 12.000 x g por 20 min a 4 °C e uma pequena pelota azul será visível na parte inferior do tubo.</li>
		<li>Retire o supernasce e lave a pelota com 850 μl de 75% de etanol gelado a 12.000 x g por 10 min a 4 °C.</li>
		<li>Remova o supernatante cuidadosamente e seque a pelota de RNA por 7-10 min, no máximo. NOTA: Certifique-se de que o RNA não é deixado por muito tempo para secar como mais-a secagem da pelota de RNA torna a solubilização muito difícil.
</li>
		<li>Uma vez seco, resuspenque a pelota de RNA em 10 μl de água livre RNAse e determine a concentração de RNA. NOTA: Determine a concentração de RNA usando um espectotômetro. Quando não estiver usando imediatamente, armazene o RNA a -80 °C.</li>
	</ol></li>
	<li>síntese de aRNA de neurônios únicos:<ol>
<li>Amplie o RNA (uma ou duas rodadas dependendo do objetivo experimental) usando sistema de amplificação linear de RNA comercialmente disponível. NOTA: O RNA total de neurônios únicos variou de ~10-60 ng. O rendimento esperado da primeira rodada de amplificação é de ~100-300 ng e a segunda rodada de amplificação é ~0,8-1,5 μg de RNA.</li>
	</ol></li>
	<li>Transcrição reversa do RNA:<ol>
<li>Para gerar cDNA a partir de aRNA, use 1,0 μg de aRNA em 20 μl de uma reação de transcrição reversa. Vários kits estão disponíveis comercialmente para este fim. NOTA: o cDNA foi sintetizado de acordo com o protocolo do fabricante usando as seguintes configurações no cicloviário térmico. 5 min a 25 °C 30 min a 42 °C 5 min a 85 °C Mantenha a 4 °C</li>
		<li>Após a etapa de transcrição reversa, armazene o cDNA a -20 °C para armazenamento a longo prazo.</li>
	</ol></li>
	<li>Design e padronização do primer: Vários excelentes programas de software estão disponíveis, gratuitamente, para projetar primers para amplificações em tempo real.<ol>
<li>Selecione 18-24 mer oligonucleotídeos que produzem amplicons de 70-110 bp e sintetize os primers usando fontes comerciais. NOTA: Dois a três pares de primer devem ser projetados para cada gene de interesse. Cada conjunto de primer precisa ser padronizado por qPCR. Os pares de primer dianteiro e reverso que foram usados para gene CREB1 (Figura 4) estão abaixo: Conjunto de primer 1 Atacante: 5'TGATTCTGACGCAAAGAAAAGA-3' Reverso: 5'-ACCGTGAGCAGAGAGTTGTAGA-3' Conjunto de primer 2 Atacante: 5'-AGGGAATGTCGATGAAAGAAGA-3' Reverso: 5'-GACACACAGGAAGTATGCCAA-3'</li>
		<li>Para selecionar o melhor primer a ser usado para as análises a jusante, monitore o valor do TC e a forma da curva de amplificação em qPCR. NOTA: Os primers que dão valores ct (limiar de ciclo) entre 10-35 na amplificação redonda de 40 rodadas e curvas suaves durante a fase exponencial do PCR seguido de um platô liso são ideais para análises de expressão genética.</li>
	</ol></li>
	<li>PCR em tempo real quantitativo (qPCR): Nota: Utilize tubos ou placas apropriados compatíveis com a máquina qPCR.<ol>
<li>Diluir cDNA para 5x com água livre de nuclease.</li>
		<li>A 2 μl de cDNA diluído, adicione 8 μl de uma mistura mestre qPCR contendo 2 μl de H2O, 5 μl de 2x SYBR Green master mix e 1,0 μl de 10 μM (cada) primer para frente e reverso.</li>
		<li>Feche os tubos e sele a placa após a pipetação cDNA e a mistura mestre. Misture o conteúdo tocando suavemente e girando a 1.500 rpm por 30 segundos.</li>
		<li>Prossiga para a configuração da máquina qPCR.</li>
	</ol></li>
</ol>

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O neurônio R15 está envolvido na regulação dos sistemas cardiovascular, digestivo, respiratório e reprodutivo30. Uma atividade de estouro regularmente rítmico do AP é uma característica do R15. Como mostrado na seção de resultados, a gravação emparelhada de R15 e L7 mostra que a preparação de gânglios preservou a atividade dos neurônios R15. Os neurônios R15 e L7 responderam apropriadamente ao Ach. Esta preparação de gânglios poderia ser mantida até 8-10 horas e a atividade eletrofisiológ...

O cérebro humano é extraordinariamente complexo com quase 100 bilhões de neurônios e trilhões de conexões sinápticas. Há quase um número igual de células nonneuronas que interagem com neurônios e regulam sua função no cérebro. Os neurônios são organizados em circuitos que regulam comportamentos específicos. Apesar dos avanços em nossa compreensão das funções cerebrais e circuitos neurais, pouco se sabe sobre a identidade dos componentes dos circuitos que controlam um comportamento específico. O conhecimento das identidades de vários componentes de um circuito facilitará muito nossa compreensão da base celular e molecular do comportamento e ajudará no desenvolvimento de novas estratégias terapêuticas para distúrbios neuropsiquiátricos.
O caracol marinho Aplysia californica tem sido um cavalo de trabalho para determinar circuitos neuronais subjacentes a comportamentos específicos1-14. O sistema nervoso da Aplysia contém aproximadamente 20.000 neurônios que são organizados em 9 gânglios diferentes. Os neurônios da Aplísia são grandes e podem ser facilmente identificados com base em seu tamanho, propriedades elétricas e posição no gânglio. A Aplysia tem um rico repertório de comportamentos que podem ser estudados. Um dos comportamentos bem estudados é o reflexo de retirada de brânquias (GWR). Os componentes centrais deste reflexo estão situados em gânglios abdominais. Componentes do circuito GWR foram mapeados e contribuições de vários componentes determinados. É importante ressaltar que o circuito GWR sofre aprendizado associativo e não associativo5,6,15-19. Décadas de estudo sobre esse reflexo também identificaram diversas vias de sinalização que têm um papel fundamental na aprendizagem e na memória20-24.
Várias preparações diferentes da Aplysia foram usadas para estudar base celular e molecular do armazenamento de memória. Estes incluem o animal intacto2,3, preparação semi-intacta1,7,13,14,16 e reconstituição dos principais componentes do circuito neural25-29. Uma preparação reduzida para explorar a Aplysia gânglios para as análises eletrofisiológicas e moleculares de circuitos neuronais identificados é descrita aqui. Foram estudados os quatro neurônios identificados a seguir. R15, um neurônio estourando, L7 e L11, dois neurônios motores diferentes e R2, um neurônio colinérgico foram estudados. R2 é o maior neurônio descrito no sistema nervoso invertebrado. Resumidamente, essa metodologia envolve o tratamento protease de gânglios, medidas eletrofisiológicas antes e depois de tratamentos farmacológicos e isolamento de neurônios únicos para análise quantitativa da expressão genética. Essa metodologia nos permite combinar análises moleculares com gravação simultânea de múltiplos neurônios. Esta metodologia foi utilizada com sucesso para estudar respostas de neurônios R15 e L7 à acetilcolina (Ach) por gravações intracelulares emparelhadas. Após medições eletrofisiológicas R15 e L7 e outros neurônios identificados como L11 e R2 foram isolados para análise quantitativa da reação em cadeia de polimerase (qPCR) da expressão do CREB1, fator de transcrição importante para o armazenamento da memória.

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			<td height="101" style="height:101px;width:120px;">Aplysia</td>
			<td style="width:123px;">National Aplysia Resource Facility, University of Miami</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">NaCl</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">S 3014-1KG</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:120px;">KCl</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">P 9333-500G</td>
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		<tr height="27">
			<td height="27" style="height:27px;width:120px;">CaCl2&#8226;2H2O</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">C5080- 500G</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">MgCl2&#8226;6H2O</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:129px;">BP 214-501</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">NaHCO4</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">S 6297-250G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">HEPES</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">H 3375-500G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Protease</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:129px;">17105-042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Trizol</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">15596-026</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Chloroform</td>
			<td style="width:123px;">MP Biomedicals</td>
			<td style="width:129px;">2194002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">100% Ethanol</td>
			<td style="width:123px;">ACROS</td>
			<td style="width:129px;">64-17-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">GlycoBlue</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9515</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">3 M NaOAc, pH 5.5</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9740</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Nuclease free water</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9737</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:120px;">MessageAmp II aRNA Amplification Kit</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM1751</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">qScript cDNA SuperMix</td>
			<td style="width:123px;">Quanta Biosciences</td>
			<td style="width:129px;">95048-100</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Power SYBR Green PCR Master Mix</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">4367659</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Forceps</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">11252-20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Scissors</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">15000-08</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:120px;">Stainless Steel Minutien Pins&#160;</td>
			<td rowspan="2" style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">26002-10 or</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:129px;">26002-20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Veriti Thermal Cycler</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">Veriti Thermal Cycler</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">5430R Centrifuge</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:129px;">5430R Centrifuge</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">7900HT Fast Real-Time PCR</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">7900HT Fast Real-Time PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:120px;">Amplifier</td>
			<td>BRAMP-01R</td>
			<td style="width:129px;">NPI Electronics</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:120px;">Digidata Converter</td>
			<td style="width:123px;">Instrutech ITC-18</td>
			<td style="width:129px;">HEKA ELEKTRONIK</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:120px;">Micro Manipulator</td>
			<td style="width:123px;">Patch Star</td>
			<td style="width:129px;">Scientifica</td>
		</tr>
	</tbody>
</table>

aplysia ganglia preparation for electrophysiological and molecular analyses of single neurons

Um grande desafio na neurobiologia é entender os fundamentos moleculares dos circuitos neurais que governam um comportamento específico. Uma vez identificados os mecanismos moleculares específicos, novas estratégias terapêuticas podem ser desenvolvidas para tratar anormalidades em comportamentos específicos causados por doenças degenerativas ou envelhecimento do sistema nervoso. O caracol marinho Aplysia californica é adequado para as investigações de base celular e molecular do comportamento porque circuitos neurais subjacentes a um comportamento específico poderiam ser facilmente determinados e os componentes individuais do circuito poderiam ser facilmente manipulados. Essas vantagens da Aplísia levaram a várias descobertas fundamentais da neurobiologia da aprendizagem e da memória. Aqui descrevemos uma preparação do sistema nervoso da Aplysia para as análises eletrofisiológicas e moleculares dos neurônios individuais. Resumidamente, o gânglio dissecado do sistema nervoso é exposto à protease para remover a baia de gânglio, de modo que os neurônios sejam expostos, mas retenham a atividade neuronal como no animal intacto. Esta preparação é usada para realizar medições eletrofisiológicas de neurônios únicos ou múltiplos. É importante ressaltar que, após o registro utilizando uma metodologia simples, os neurônios poderiam ser isolados diretamente dos gânglios para análise de expressão genética. Esses protocolos foram utilizados para realizar gravações eletrofisiológicas simultâneas de neurônios L7 e R15, estudar sua resposta à acetilcolina e à expressão quantitante do gene CREB1 em neurônios isolados L7, L11, R15 e R2 da Aplysia.

Os pesos dos animais utilizados neste estudo variaram de 100 a 200 g. Seguindo os protocolos descritos, realizamos medições eletrofisiológicas e análise molecular de neurônios de gânglios abdominais isolados de animais que variam de 2-5 g a 200-300 g.
A padronização do tratamento protease é importante para medições eletrofisiológicas bem sucedidas de neurônios nos gânglios. Inicialmente, foram utilizadas concentrações e durações de protease múltipla (Dispase) e potenciais de...

Watch this Scientific Journal Video about Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons at JoVE.com

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A caracol marinha Aplysia californica tem sido amplamente utilizada como modelo de neurobiologia para os estudos sobre base celular e molecular do comportamento. Aqui é descrita uma metodologia para explorar o sistema nervoso da Aplysia para as análises eletrofisiológicas e moleculares de neurônios únicos de circuitos neurais identificados.

Aplysia Preparação de Ganglia para Análises Eletrofisiológicas e Moleculares de Neurônios Únicos

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

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Research

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Bioengineering

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

9.0K visualizações.  The Scripps Research Institute, Florida. A caracol marinha Aplysia californica tem sido amplamente utilizada como modelo de neurobiologia para os estudos sobre base celular e molecular do comportamento. Aqui é descrita uma metodologia para explorar o sistema nervoso da Aplysia para as análises eletrofisiológicas e moleculares de neurônios únicos de circuitos neurais identificados.

Vídeo: Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

A Armadilha hidrodinâmica microfluídicos baseados em partículas individuais

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Bioengenharia

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Avaliação de Biomateriais para ampliação vesical utilizando análises cistométricos Em modelos de roedores Vários

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

Entanglement Molecular e Electrospinnability de Biopolímeros

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

O tecido triturado em Compressed Collagen: Um BioTransplant contendo celular para o reparo Reconstructive Single-encenado

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

A técnica de livre-Label para a imagem espaço-temporal do Secreções única célula

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Preparação de Silicon Nanowire transistor de efeito de campo para aplicações químicas e Biossensoriais

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Preparação de Matriz Extracelular fibras de proteína de Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Reconhecimento Molecular de engenharia com Polymers Bio-mimético em únicos Walled nanotubos de carbono

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Dissecação, digitalização MicroCT e morfométricas Análises do Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Preparação, purificação e utilização de lipossomas contendo ácido graxo

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...