Name: retroductal nanoparticle injection to the murine submandibular gland
Uploaded: 2018-05-03T14:15:00
Description: Watch this Scientific Journal Video about Retroductal Nanoparticle Injection to the Murine Submandibular Gland at JoVE.com

Pesquisa reportada nesta publicação foi apoiada pelo Instituto Nacional de dentária e Craniofacial Research (NIDCR) e o National Cancer Institute (NCI) do institutos nacionais da saúde sob prêmio número R56 DE025098, DE027695 UG3 e CA206296 F30. O conteúdo é exclusivamente da responsabilidade dos autores e não representa necessariamente a opinião oficial do institutos nacionais da saúde. Este trabalho também foi apoiado pela NSF DMR 1206219 e a inovação IADR no prêmio de cuidado Oral (2016).
Gostaríamos de agradecer sua ajuda na realização de experimentos IVIS Jayne Gavrity. Gostaríamos de agradecer a Karen Bentley para sua entrada e assistência na realização de EM. Gostaríamos de agradecer sua ajuda com IHC Pei-Lun Weng. Gostaríamos de agradecer a Matthew Ingalls por sua assistência na preparação de figura. Gostaríamos de agradecer o Dr. Elaine Smolock e Emily Wu para leitura crítica deste manuscrito.

Todos na vivo os procedimentos descritos abaixo foram aprovados pela Comissão Universidade recursos animais na Universidade de Rochester, Rochester, NY.
1. preparação
<ol><li>Usando o tubo de cateter intracraniano 32G com inserir fio, corte 3 cm do tubo para formar uma ponta chanfrada, aproximadamente 45° ao eixo longo. Confirme que o fio é mais do que o tubo de pelo menos 1 cm.</li>
	<li>Carrega 50 µ l de solução de nanopartículas PSMA (Figura 1</stron...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy-induced+salivary+dysfunction:+Structural+changes,+pathogenetic+mechanisms+and+therapies.">Radiotherapy-induced salivary dysfunction: Structural changes, pathogenetic mechanisms and therapies.</a> Archives of Oral Biology. 60 (12), 1802-1810 (2015).</li><li>Dirix, P., Nuyts, S., Vander Poorten, V., Delaere, P., Van den Bogaert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+xerostomia+after+radiotherapy+on+quality+of+life.">The influence of xerostomia after radiotherapy on quality of life.</a> Supportive Care in Cancer. 16 (2), 171-179 (2008).</li><li>Vissink, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+management+of+salivary+gland+hypofunction+and+xerostomia+in+head-and-neck+cancer+patients:+successes+and+barriers.">Clinical management of salivary gland hypofunction and xerostomia in head-and-neck cancer patients: successes and barriers.</a> International Journal of Radiation Oncology Biology Physics. 78 (4), 983-991 (2010).</li><li>Delporte, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+fluid+secretion+after+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+to+irradiated+rat+salivary+glands.">Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands.</a> Proceedings of the National Academy of Sciences. 94 (7), 3268-3273 (1997).</li><li>Samuni, Y., Baum, B. 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D., Mezey, E., Key, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersed+donor+salivary+gland+cells+are+widely+distributed+in+the+recipient+gland+when+infused+up+the+ductal+tree.">Dispersed donor salivary gland cells are widely distributed in the recipient gland when infused up the ductal tree.</a> Biotechnic &amp; Histochemistry. 84 (6), 253-260 (2009).</li><li>Grundmann, O., Fillinger, J. L., Victory, K. R., Burd, R., Limesand, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+radiation+therapy-induced+salivary+gland+dysfunction+in+mice+by+post+therapy+IGF-1+administration.">Restoration of radiation therapy-induced salivary gland dysfunction in mice by post therapy IGF-1 administration.</a> BMC Cancer. 10, 417-417 (2010).</li><li>Limesand, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-Like+Growth+Factor-1+Preserves+Salivary+Gland+Function+After+Fractionated+Radiation.">Insulin-Like Growth Factor-1 Preserves Salivary Gland Function After Fractionated Radiation.</a> International Journal of Radiation Oncology Biology Physics. 78 (2), 579-586 (2010).</li><li>Marmary, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+loss+of+salivary+gland+function+is+driven+by+cellular+senescence+and+prevented+by+IL-6+modulation.">Radiation-induced loss of salivary gland function is driven by cellular senescence and prevented by IL-6 modulation.</a> Cancer Research. , (2016).</li><li>Baum, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+responses+to+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+for+radiation-induced+salivary+hypofunction.">Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (47), 19403-19407 (2012).</li><li>Arany, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-apoptotic+gene+knockdown+mediated+by+nanocomplexed+siRNA+reduces+radiation+damage+in+primary+salivary+gland+cultures.">Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures.</a> Journal of Cellular Biochemistry. 113 (6), 1955-1965 (2012).</li><li>Benoit, D. S. W., Henry, S. M., Shubin, A. D., Hoffman, A. S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-responsive+polymeric+siRNA+carriers+sensitize+multidrug+resistant+ovarian+cancer+cells+to+doxorubicin+via+knockdown+of+polo-like+kinase+1.">pH-responsive polymeric siRNA carriers sensitize multidrug resistant ovarian cancer cells to doxorubicin via knockdown of polo-like kinase 1.</a> Molecular pharmaceutics. 7 (2), 442-455 (2010).</li><li>Malcolm, D. W., Varghese, J. J., Sorrells, J. E., Ovitt, C. E., Benoit, D. S. 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Retroductal injeção é fundamental para a entrega de drogas localizadas à glândula salivar. Essa técnica tem aplicações na triagem de agentes terapêuticos para condições, incluindo a síndrome de Sjogren e RIH9,10,28. Entrega da droga direto para a SMG através da injeção de retroductal fornece uma vantagem chave sobre administração sistémica no seu potencial para reduzir os efeitos fora do alvo, incluindo ativaç...

Disfunção da glândula salivar tem várias etiologias, incluindo a síndrome de Sjögren, uma auto-imune mediada por perda de tecido secretor funcional e radiação induzida hipossalivação (RIH), uma comum sequelas de cabeça e pescoço câncer radioterapia1. Perda da função salivar devido qualquer condição predispõe indivíduos a infecção oral e sistêmica, cárie dentária, disfunção digestiva e deglutição, Imparidade de discurso e depressão1,2,3. Como resultado, qualidade de vida significativamente sofre, com intervenções limitadas a paliação dos sintomas ao invés de cura4. Para investigar novas terapias na vivo, é de interesse para administrar compostos bioativos diretamente à glândula salivar.
Injeção de Retroductal é um método valioso para entregar compostos bioativos diretamente para as glândulas salivares e testar a eficácia na doença, lesão, ou sob a homeostase do tecido normal. As três principais glândulas salivares são as parótidas (PG), a submandibular (SMG) e a sublingual (SLG), todos que vazio na cavidade oral através de ductos excretores. A anatomia do murino SMG permite acesso direto através de cateterização do ducto de Wharton, localizado no assoalho da boca sob a língua5. Após a cateterização, solvated drogas podem ser administradas diretamente para a SMG. Após o parto retroductal, difusão extra glandular é restrito pela cápsula de tecido circundante, que regula a troca de material com em torno de estruturas6. O SMG e seu ducto estruturam-se da mesma forma em seres humanos e rotineiramente são acessados durante SMG cirurgia e sialoendoscopy7. Em humanos e ratos, o PG é igualmente acessível através do duto de Stensen na mucosa bucal8.
Em modelos murino da RIH, injeção de retroductal SMG tem sido utilizada para entregar terapêutica incluindo fatores de crescimento, as células primárias, vetores adenovírus, citocinas e compostos antioxidantes modular a resposta celular a lesões, e reduzir o tecido danos5,9,10,11,12,13,14,15,16. O mais notável sucesso clínico da injeção de retroductal é a administração do vetor adenovírus para direcionar a expressão de um canal de água (aquaporina 1; AQP1) em pacientes após a radiação para câncer de cabeça e pescoço17.
Anteriormente, desenvolvemos e demonstrado a eficácia de um sistema de nanopartículas poliméricas-siRNA retroductally injetado para proteger a função da glândula salivar de RIH11,18,19,20. Como uma extensão de nossos trabalhos anteriores, aqui, demonstramos nosso protocolo para injeção de SMG retroductal usando uma nanopartícula fluorescente etiquetada (NP) capaz de carregar e entregar outra forma pouco solúvel drogas21,22, 23.
Podemos ter sintetizado o NP de um copolímero de diblock composto de poli (estireno-alt-hidrazida anhydride)-b-poly(styrene) (PSMA) através da polimerização de fragmentação (balsa) de cadeia de adição reversível, conforme descrito anteriormente,21. Através da troca de solvente, estes polímeros espontaneamente auto-montagem em estruturas de NP micelle com um hidrofóbico hidrofílico e interior exterior21. O NPs é rotulado com o fluoróforo Texas-vermelho para permitir a verificação da entrega NP nas glândulas sem sacrificar o animal. Viver a imagem animal e imuno-histoquímica SMG é mostrado em 1 h e 1 dia após a injeção.
Esta actualizado e protocolo de canulação reprodutível deve permitir que outros conseguir injeção de retroductal. Esperamos que esta técnica refinada se torne crítica para estudos em vivo e desenvolvimento terapêutico24,25.

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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Pilocarpine hydrochloride</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P6503</td>
			<td style="width:225px;">Pilocarpine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Student Vannas Spring Scissors</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">91500-9</td>
			<td>Spring Scissors for Tracheostomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Sterile Saline Solution</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">RDI30296H</td>
			<td>Saline</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:219px;">Dumont #7 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11274-20</td>
			<td>Curved Forceps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:219px;">Dumont #5 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11251-10</td>
			<td>Straight Forceps</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:219px;">Standard Pattern Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11000-12</td>
			<td>Blunt Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Fine Scissors- Tungsten Carbide</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">14568-09</td>
			<td>Dissection Scissors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Microhematocrit Heparinized Capillary Tubes</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:119px;">22362566</td>
			<td>Capillary tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Lubricant Eye Ointment</td>
			<td style="width:128px;">Refresh</td>
			<td style="width:119px;">N/A</td>
			<td>Refresh Lacri-Lube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Goat polyclonal anti-Nkcc1</td>
			<td style="width:128px;">Santa Cruz Biotech</td>
			<td style="width:119px;">SC-21545</td>
			<td>Nkcc1 Antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">GraphPad Prism</td>
			<td style="width:128px;">GraphPad</td>
			<td style="width:119px;">ver6.0</td>
			<td>Statistical Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Cotton tipped applicator</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td>Applicator for eye ointment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">0.5cc Insulin Syringe, 29G x 1/2&#34;</td>
			<td style="width:128px;">BD</td>
			<td style="width:119px;">7629</td>
			<td>Syringe for intraperitoneal injection</td>
		</tr>
	</tbody>
</table>

retroductal nanoparticle injection to the murine submandibular gland

Dois objetivos comuns das glândulas salivares terapêutica são prevenção e cura da disfunção do tecido seguindo ou auto-imune ou radiação. Entregando localmente compostos bioativos para as glândulas salivares, maiores concentrações de tecido podem ser alcançadas com segurança contra administração sistémica. Além disso, fora o tecido-alvo os efeitos da acumulação extra glandular de material podem ser drasticamente reduzidos. A este respeito, injeção de retroductal é um método amplamente usado para investigar a biologia das glândulas salivares e a fisiopatologia. Retroductal administração de fatores de crescimento, as células primárias, vetores adenovírus e drogas pequena molécula mostrou para apoiar a função da glândula no cenário do ferimento. Nós anteriormente demonstraram a eficácia de uma estratégia de siRNA nanoparticle retroductally injetada para manter a função da glândula após irradiação. Aqui, um método altamente eficaz e reprodutível para administrar nanomateriais para a glândula submandibular murino através do ducto de Wharton é Detalhado (Figura 1). Descrevemos a acessar a cavidade oral e descrevem as etapas necessárias ao duto de cannulate da Wharton, com novas observações servindo como verificações de qualidade durante todo o procedimento.

Injeção de Retroductal pode ser usada para administrar o NPs para a SMG murino (Figura 1). Aqui, nós entregamos a 50 µ g PSMA NPs rotulado com vermelho Texas fluoróforo.
Colocação adequada do mouse permite fácil acesso e visualização do assoalho da boca (Fig. 2A-B). As papilas submandibulares são identificadas como duas saliências carnudas d...

Watch this Scientific Journal Video about Retroductal Nanoparticle Injection to the Murine Submandibular Gland at JoVE.com

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Entrega de drogas local para as glândulas submandibulares é de interesse em biologia de glândula salivar de entendimento e para o desenvolvimento de novas terapêuticas. Apresentamos um protocolo de injeção retroductal atualizadas e detalhadas, destinado a melhorar a precisão de entrega e reprodutibilidade experimental. O aplicativo aqui apresentado é a entrega de nanopartículas poliméricas.

Injeção de Retroductal de nanopartículas para a glândula Submandibular murino

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

The overall goal of this procedure is to administer nanomaterials directly to the murine submandibular gland. The main advantage of this technique is that it achieves direct delivery of compounds to the submandibular gland while bypassing the systemic circulation. The implications of this technique extend toward therapy of salivary gland disfunction following radiation treatment for head and neck malignancy.Generally, individuals new to this method may be challenged by locating and cannulating the submandibular duct while the animal is sedated. To begin this procedure, cut three centimeters of a 32 gauge intracranial catheter tubing with wire inset to form a beveled end approximately 45 degrees to the long axis. Confirm that the wire is at least one centimeter longer than the tubing.Creating a beveled edge to the tubing will ease ductal cannulation. If the tubing gets bent during the procedure, a fresh beveled edge should be cut. Next, load 50 microliters of PSMA nanoparticle solution or other injection material into a Hamilton syringe.To reduce the probability of burrow trauma during injection, attach the catheter tubing to the syringe and expel dead volume. Inspect the injection solution to ensure the nanoparticle is fully solvated to prevent ductal obstruction. Then prepare atropine solution at 0.1 milligram per milliliter.To visualize the ductal entry point, place an anesthetized mouse in the prone position on a custom stage. Then apply ointment to the eyes to prevent dryness during the procedure. Afterward, secure the maxillary incisors over a metal beam and use an elastic band to apply downward tension behind the mandibular incisors.Align the mouse beneath the dissecting microscope to visualize the base of the jaw. To widen the mouth, use a custom curved steel retractor to apply tension to the buccal mucosa bilaterally. To visualize the submandibular papilla, grasp and gently lift the tongue from the floor of the mouth using blunt forceps.Place a piece of cotton between the tongue and the buccal mucosa to enable further manipulation within the oral cavity. To perform ductal cannulation and line placement, grasp the catheter tubing with a wire inset. For optimum manual control during cannulation, align the tubing with the curvature of the forceps.Using the dissecting microscope, move the forceps and wire into the field of view. Using the wire inset, gently apply pressure into the base of one submandibular papilla to produce a small superficial mucosal puncture that will facilitate later entry of the catheter tubing. If resistance is encountered, cut fresh beveled tips on both the tubing and wire inset with sharp dissecting scissors.Following the entry, withdraw the stylet, then, using the dissecting microscope, confirm the presence of saliva at the puncture site. Subsequently, retract the stylet further within the tubing. Avoid forceful or sudden movement of the stylet that may cause bleeding or compromise ductal integrity.To ensure that injection tubing will fit into the Wharton's duct opening, insert tubing containing the stylet as a rigid guide into the previously made puncture. To prevent back pressure from prolonged to ductal obstruction, withdraw the tubing. Under the microscope, ensure that an opening can be seen in the submandibular papilla.If visible bleeding occurs, remove the stylet and reattempt the procedures on the opposing submandibular papilla. Next, without moving the mouse, administer one milligram per kilogram atropine solution intraperitoneally to reduce elevation during the procedure. Then, wait for five to 10 minutes.Under the dissecting microscope, insert the syringe tubing into the orifice. If resistance is encountered, cut a fresh beveled end on the tubing and reattempt it. Once the tubing is in place within the submandibular papilla, slowly advance three to five millimeters into the duct.Then, release the tubing from the forceps. To improve the seal between the tubing and the submandibular papilla, dry the interface by gently blotting with gauze for one minute. Inspect to confirm that the position of the tubing has not shifted during drying.In this procedure, inject the material at a rate of 10 microliters per minute. Ensure that the mouse remains sedated and is not showing signs of distress. Following the injection, maintain syringe pressure for five minutes to improve the retention of material within the Wharton's duct and the submandibular gland.Inspect the submandibular papilla periodically to ensure that tubing does not exit the ductal orifice. Maintaining injection pressure will enhance the effectiveness of retroductal delivery. Using fine forceps, grasp and gently and withdraw the tubing from the submandibular papilla.Remove the retractor and cotton from the oral cavity before moving the mouse away from the stage. To verify nanoparticle delivery, in vivo imaging system shows the lateralization of red fluorescent signal to the treated side of the mouse one hour post injection. And this image shows that the nanoparticle signal has decreased significantly 24 hours after injection.To confirm nanoparticle persistence in the submandibular glands 24 hours following injection, they were sectioned and viewed by fluorescent imaging. Shown here are the uninjected control submandibular glands stained for aquaporin 5 and keritin five marking the secretory acini and ductal cells respectively. In retroductal nanoparticle injected submandibular glands, aquaporin five and keritin five stains show normal gland morphology and nanoparticles taken up in both acini and ducts.Once mastered this technique can be done in 20 minutes if it is performed properly. While attempting this procedure, it's important to remember to avoid any extraneous movements that could dislodge the cannula or disrupt the seal made between the tubing and the submandibular papilla. Following the procedure, other methods like immunostaining, RNA extraction, or flow cytometry can be performed in order to evaluate gene expression or nanomaterial localization in the submandibular gland.After its development, this technique paved the way for salivary gland researchers to screen and test compounds for xerostomia prevention. After watching this video, you should have a good understanding of how to administer nanomaterials directly to the murine submandibular gland via cannulation of Wharton's duct.

retroductal-nanoparticle-injection-to-the-murine-submandibular-gland

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Bioengenharia

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Síntese, Assembléia e Caracterização de monocamada Protected Films nanopartículas de ouro para Eletroquímica monocamada Protein

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Um modelo de cultura 3D Hormone-responsive da Human glândula mamária Epitélio

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Determinação do Potencial Zeta de nanopartículas de translocação através de velocidades por meio de um Tunable Nanopore: Utilizando partículas modificadas-ADN como um Exemplo

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Entrega Intraductal ao Coelho glândula mamária

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Um sistema integrado para acionar remotamente a transdução do sinal intracelular por Photoactivation Upconversion Nanoparticle-mediada da quinase

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Microscopia de fluorescência de luz-folha para o estudo do coração murino

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

Protocolo de injeção de lipidico para medições de cristalografia serial no Síncrotron Australiano

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Transplante de um Patch Bioimpresso 3D em um modelo murino de infarto do miocárdio

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

Melhorando a reprodutibilidade para atender informações mínimas para estudos de diretrizes extracelulares de vesículas 2018 em análise de rastreamento de nanopartículas

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...